Single-cell analysis reveals that cryptic prophage protease LfgB protects Escherichia coli during oxidative stress by cleaving antitoxin MqsA

ABSTRACT Although toxin/antitoxin (TA) systems are ubiquitous, beyond phage inhibition and mobile element stabilization, their role in host metabolism is obscure. One of the best-characterized TA systems is MqsR/MqsA of Escherichia coli, which has been linked previously to protecting gastrointestinal species during the stress it encounters from the bile salt deoxycholate as it colonizes humans. However, some recent whole-population studies have challenged the role of toxins such as MqsR in bacterial physiology since the mqsRA locus is induced over a hundred-fold during stress, but a phenotype was not found upon its deletion. Here, we investigate further the role of MqsR/MqsA by utilizing single cells and demonstrate that upon oxidative stress, the TA system MqsR/MqsA has a heterogeneous effect on the transcriptome of single cells. Furthermore, we discovered that MqsR activation leads to induction of the poorly characterized yfjXY ypjJ yfjZF operon of cryptic prophage CP4-57. Moreover, deletion of yfjY makes the cells sensitive to H2O2, acid, and heat stress, and this phenotype was complemented. Hence, we recommend yfjY be renamed to lfgB (less fatality gene B). Critically, MqsA represses lfgB by binding the operon promoter, and LfgB is a protease that degrades MqsA to derepress rpoS and facilitate the stress response. Therefore, the MqsR/MqsA TA system facilitates the stress response through cryptic phage protease LfgB. IMPORTANCE The roles of toxin/antitoxin systems in cell physiology are few and include phage inhibition and stabilization of genetic elements; yet, to date, there are no single-transcriptome studies for toxin/antitoxin systems and few insights for prokaryotes from this novel technique. Therefore, our results with this technique are important since we discover and characterize a cryptic prophage protease that is regulated by the MqsR/MqsA toxin/antitoxin system in order to regulate the host response to oxidative stress.

1) Have the authors done any investigations in an rpoS negative background?This data would greatly strengthen the experimental interpretation.
2) The authors encountered some issues with the low solubility of the LfgB protein.Might one possible explanation be that this protein is membrane-associated?
3) In lines 144-145, the authors make the claim that cryptic prophages are beneficial and involved in the stress response.I would strongly recommend softening this claim since the authors have investigated a subset of prophages and not all prophages.
4) As a minor point, please insert "(TA)" without the quotation marks following "Toxin/antitoxin" in the opening sentence of the introduction.That will nicely define this acronym.
Reviewer #2 (Comments for the Author): The manuscript by Fernández-García describes the possible role of the MqsRA toxin-antitoxin system and the LfgB protease of CP4-57 cryptic prophage in response to oxidative stress in E. coli.They first show that the mqsRA deletion mutant is more sensitive to oxidative stress than the wild type strain, and identified several genes that are induced in the wild type strain when compared to the mqsRA mutant following H2O2 exposure.Among these, genes of the lfgABCDE operon of the cryptic prophage CP4-57 are the focus of this study.They authors show that both lfgA and lfgB mutations prevented cells from surviving oxidative stress, and that the lfgB mutant had a reduced catalase activity and was more sensitive heat and acid treatments.The link between MqsRA and the putative LfgB protease was further investigated, showing that MqsA can bind and repress transcription of the lfg operon.Based on the possible degradation of MqsA by LfgB observed in vitro, the authors conclude that LfgB controls the oxidative stress response through MqsA degradation.Although the model is interesting, it is not supported by the data presented.Comments: -The main conclusion that MqsA is a substrate of the putative LfgB protease in vitro is not supported by the data and there is no evidence showing that MqsA is accumulating in the absence of lfgB in vivo.The important in vitro experiments from Figure 2 lack all the controls and does not show proper kinetics of degradation under reasonable experimental conditions.How can the authors be confident that LfgB, but not any other proteases from the actual purification, degrades MqsA?How is an overnight incubation at 37{degree sign}C relevant in this case?How were the MqsA degradation products identified?Such critical issues should have been properly addressed.
-Complementation of the H2O2 sensitivity of the mqsRA mutant was not performed.
-There is no evidence that the lfgB transcript is cleaved by MqsR.

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Spectrum03471-23: Single-Cell Analysis Reveals Cryptic Prophage Protease LfgB Protects Escherichia coli During Oxidative Stress by Cleaving Antitoxin MqsA
We wish to thank the editor and two reviewers for their help with this manuscript and feel the manuscript is improved due to the careful review.We have addressed all the issues as indicated below (our comments are underlined and the changed text is highlighted yellow in the revised manuscript).Note the line numbers below refer to the revised text.Specifically, we have conducted 5 additional experiments to (i) determine the impact of H2O2 and MqsRA on yfjX transcription via qRT-PCR for BW25113 vs. BW25113 mqsRA ∆Kan using population averages rather than single cells and found the changes in yfjX transcription can only be detected via the single-cell approach (results added to line 90 and raw data in Table S2A),(ii) determine the impact of LfgB production on rpoS + transcription via qRT-PCR, which shows 3X induction rpoS + when LfgB is produced due to likely degradation of MqsA and the derepression of rpoS + (Table S2C, line 148), (iii) determine the impact of LfgB on catalase activity in the absence of RpoS and found LfgB, as expected, has less effect in the absence of RpoS (Fig 1 and line 151), (iv) add purified Lon and alpha-caesin/BSA as positive controls for the protein gel (Fig. S6), and (v) use mass spectrometry to show the confirm LfgB was purified correctly (Fig. S7).
We also improved the main figures by consolidating the results and by adding the catalase results to Fig. 1 and re-drew the mechanism schematic of Fig. 2 to increase clarity.

Reviewer 1
This is a very interesting and timely study in which the authors have identified a new role for a toxin-antitoxin system during E. coli growth and its influence in regulating oxidative stress.There are a number of minor questions that I would like the authors to address: 1) Have the authors done any investigations in an rpoS negative background?This data would greatly strengthen the experimental interpretation.
As suggested, we measured investigated the effect of LfgB on catalase activity in the absence of RpoS by producing LfgB via BW25113 rpoS/pCA24N-lfgB vs. BW25113/pCA24N-LfgB and found, as expected, that in the absence of RpoS, LfgB is less effective in increasing catalase.This is most likely due to the fact that there is no activation of RpoS by LfgB degrading MqsA, which represses rpoS (Fig. 1A, line 151) 2) The authors encountered some issues with the low solubility of the LfgB protein.Might one possible explanation be that this protein is membrane-associated?
As suggested, we have added your idea to the manuscript (line 146) 3) In lines 144-145, the authors make the claim that cryptic prophages are beneficial and involved in the stress response.I would strongly recommend softening this claim since the authors have investigated a subset of prophages and not all prophages.
As suggested, to strengthen this claim, we now refer to our three additional, earlier studies in which we (i) deleted all 9 E. coli cryptic prophages (166 kb, so we have studied all 9 E. coli cryptic prophages) to show cryptic prophages increase environmental fitness (Nature Commun 2010, cited over 600 times) and also refer to results from this 2010 manuscript that show the same cryptic prophage in this current paper that encodes LfgB, CP4-57, increases fitness for acid stress by 54fold 4 , (ii) found that, although cryptic prophages increase environmental fitness, their residual lytic capabilities must be silenced by CRISPR-Cas 2 , and (iii) discovered CP4-57 helps the cell resuscitate from the persister state by monitoring phosphate concentrations through regulator AlpA 1 .(line 165).4) As a minor point, please insert "(TA)" without the quotation marks following "Toxin/antitoxin" in the opening sentence of the introduction.That will nicely define this acronym.
As suggested, we have defined the acronym here (line 25), and it was previously defined in the Abstract (line 2).

Reviewer 2
The manuscript by Fernández-García describes the possible role of the MqsRA toxin-antitoxin system and the LfgB protease of CP4-57 cryptic prophage in response to oxidative stress in E. coli.They first show that the mqsRA deletion mutant is more sensitive to oxidative stress than the wild type strain, and identified several genes that are induced in the wild type strain when compared to the mqsRA mutant following H2O2 exposure.Among these, genes of the lfgABCDE operon of the cryptic prophage CP4-57 are the focus of this study.They authors show that both lfgA and lfgB mutations prevented cells from surviving oxidative stress, and that the lfgB mutant had a reduced catalase activity and was more sensitive heat and acid treatments.The link between MqsRA and the putative LfgB protease was further investigated, showing that MqsA can bind and repress transcription of the lfg operon.Based on the possible degradation of MqsA by LfgB observed in vitro, the authors conclude that LfgB controls the oxidative stress response through MqsA degradation.
Although the model is interesting, it is not supported by the data presented.
The reviewer is, of course, correct, and so we strive now to indicate more clearly which parts of the proposed mechanism are speculative (e.g., lines 135 by adding "likely" and line 141, "…we cannot strictly rule out…").However, this is the first use of single-cell transcriptomics for the TA field and one of the first single-cell transcriptomic studies that actually provides some new biological insight (all reports to date have just confirmed existing, whole population physiology).Moreover, we have conducted new experiments that corroborate our results by showing, via the new PCR study, that we cannot detect changes in lfgB in population-averaged cells, and so, single-cell transcriptomics are necessary for link MqsRA to previouslyuncharacterized lfgB (line 90).
Therefore, herein, we (i) derived from single-cell analysis one of the first, if not the first, physiologically-relevant new insights for this nascent method for procaryotes, (ii) applied single-cell analysis to TA systems for the first time, (iii) studied the wild-type and deletion mutant so no TA overexpression is involved, (iv) discovered and characterized an operon in a cryptic prophage that is used by the host in its oxidative stress response, and (v) found a physiological role for the type II TAs, MqsR//MqsA (H2O2 stress response) when most studies fail to find a physiological role (other than phage inhibition, which we discovered first in 1996), and prominent labs (e.g.Laub and Van Melderen) incorrectly have concluded there is not expression for these ubiquitous TAs (except the Laub lab has shown the Van Melderen lab was incorrect, at least in the incorrect lack of promoter activity for mqsRA and the Laub las has contradicted itself by finding the physiological role of MqsRAC for phage inhibition where it claimed no protein was produced previously with this TAs).Hence, our results will have a dramatic impact on the TA field by demonstrating clearly a role in cell physiology distinct from phage inhibition as well as revealing more about how the tools of the former enemy of the cell, cryptic prophages, are incorporated into the host stress response.So although we have not fully elucidated the mechanism, we think that our accomplishments are considerable for a single paper.

Comments:
1.The main conclusion that MqsA is a substrate of the putative LfgB protease in vitro is not supported by the data and there is no evidence showing that MqsA is accumulating in the absence of lfgB in vivo.a.As suggested, we added 'likely' through MqsA degradation (line 135) since our protease work is imperfect due to poor LfgB activity (although we have tried repeatedly and with protein tags); however, we do demonstrate some degradation of MqsA in the presence of purified LfgB in Fig. 2 and have now added both mass spectroscopy data to confirm LfgB was purified and SDS-PAGE controls as indicated below.
b.The suggested experiment of quantifying MqsA accumulation in single cells is nearly impossible, i.e., showing MqsA accumulation in a lfgB mutant, as that would require technology to see MqsA levels in single cells and to our knowledge, single cell proteomics is not that developed yet.Moreover, we also feel this is unnecessary since the whole point of this manuscript is to discover new biology from gene expression in single cells, and we did that successfully (probably for the first time in any lab and certainly the first time for toxin/antitoxin systems).
The important in vitro experiments from Figure 2 lack all the controls and does not show proper kinetics of degradation under reasonable experimental conditions.c.As suggested, along with showing previously MqsA in the absence of protease LfgB, we now show LfgB has protease activity on α-casein and BSA as well as use purified Lon protease as positive control with α-casein and BSA (Fig. S6).We recognize these protein gels are rough, but degradation can be seen by seeing the reduction of the α-casein and BSA bands with both Lon and LfgB.
2. How can the authors be confident that LfgB, but not any other proteases from the actual purification, degrades MqsA?
The reviewer is correct but the experiments required for this control are beyond the scope of this manuscript.Therefore, we added to the manuscript "although we cannot strictly rule out that other proteases are present in the purified LgfB purification."(line 141).
In addition, we now show via mass spectrometry data that LfgB was purified correctly as it clearly shows we purified LfgB well and clearly shows its degradation, likely due to self-digestion (Fig. S7).These data rule out nearly completely that background proteases are responsible for the degradation of MqsA seen in Fig. 1D.(line 144).
3. How is an overnight incubation at 37{degree sign}C relevant in this case?
As suggested, we now indicate now indicate the LfgB may be membrane-associated and that is why we do not see robust protease activity.(line 146) 4. How were the MqsA degradation products identified?Such critical issues should have been properly addressed.
Unfortunately, we were unable to assay MqsA degradation products; however, we were able to use mass spectrometry to determine how protease LfgB degrades itself and have added these results to Fig. S7.
5. Complementation of the H2O2 sensitivity of the mqsRA mutant was not performed.
We note that complementation of the lfgB mutation for the H2O2 phenotype was made (see Fig. S2) since LfgB is the primary focus of this manuscript.Complementation of the mqsRA mutant H2O2 and acid phenotypes along with data showing production of MqsA reduces peroxidase activity is not necessary here as we previously published this in Fig. 2bcd of Wang et al. 2011 3 , and we now indicate that in the text (line 76).
6.There is no evidence that the lfgB transcript is cleaved by MqsR.
We agree and that is why we already qualified our statement as "MqsR may degrade the mRNA containing lfgB" (line 122), and we also do not show MqsR cleaving the lfgB mRNA in the schematic (Fig. 2).We merely tried to indicate where MqsR could possibly cleave lfgB mRNA by identifying likely single-stranded regions (Fig. S3).Many thanks for addressing the comments of both reviewers to their full saturation.It was a pleasure reading the revised manuscript.
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