A mobile genetic element increases bacterial host fitness by manipulating development
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Curated by eLife
Evaluation Summary:
All the reviewers were in agreement that this is an exceptionally rigorous paper that sets an important precedent for how mobile genetic elements can influence host biology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
Horizontal gene transfer is a major force in bacterial evolution. Mobile genetic elements are responsible for much of horizontal gene transfer and also carry beneficial cargo genes. Uncovering strategies used by mobile genetic elements to benefit host cells is crucial for understanding their stability and spread in populations. We describe a benefit that ICE Bs1 , an integrative and conjugative element of Bacillus subtilis , provides to its host cells. Activation of ICE Bs1 conferred a frequency-dependent selective advantage to host cells during two different developmental processes: biofilm formation and sporulation. These benefits were due to inhibition of biofilm-associated gene expression and delayed sporulation by ICE Bs1 -containing cells, enabling them to exploit their neighbors and grow more prior to development. A single ICE Bs1 gene, devI (formerly ydcO ), was both necessary and sufficient for inhibition of development. Manipulation of host developmental programs allows ICE Bs1 to increase host fitness, thereby increasing propagation of the element.
Article activity feed
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Response to Reviewer #2 (Public Review):
Due to the regulation of devI by cell-cell signaling (rapI-phrI), biofilm formation and sporulation should proceed normally in a clonal population of ICEBs1-containing cells. We show in Figure 3 (previously figure 2) that when the ICEBs1-containing cells make up the majority of a co-culture (90%), that the timing of sporulation is similar to cells without ICEBs1. When all (or virtually all) cells in a clonal population contain ICEBs1 the element is not activated. The function of the RapI-PhrI system is to 1) recognize the presence of neighboring cells of the same species due to cell-cell signaling activating transcription of rapI; and 2) recognize whether or not those neighboring cells already contain a copy of ICEBs1, by virtue of accumulation of the peptide PhrI, which then inhibits the …
Response to Reviewer #2 (Public Review):
Due to the regulation of devI by cell-cell signaling (rapI-phrI), biofilm formation and sporulation should proceed normally in a clonal population of ICEBs1-containing cells. We show in Figure 3 (previously figure 2) that when the ICEBs1-containing cells make up the majority of a co-culture (90%), that the timing of sporulation is similar to cells without ICEBs1. When all (or virtually all) cells in a clonal population contain ICEBs1 the element is not activated. The function of the RapI-PhrI system is to 1) recognize the presence of neighboring cells of the same species due to cell-cell signaling activating transcription of rapI; and 2) recognize whether or not those neighboring cells already contain a copy of ICEBs1, by virtue of accumulation of the peptide PhrI, which then inhibits the activity of RapI, thereby preventing de-repression of Pxis.
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Response to Reviewer #1 (Public Review):
Suggestions for improvement:
The authors show that the gene devI is necessary and sufficient for ICE-mediated delay of development initiation. Gene expression analyses suggest this delay affects the earliest stages of development (genes under control of spo0A, the master regulator of sporulation, are affected). I think the authors could investigate the mechanism of spo0A inhibition in more detail. Which aspect of spo0A function is affected by DevI? Starvation sensing, spo0A expression, activation of upstream kinases (KinA?), phosphorelay, or binding of Spo0A~P to promoters?
Excellent questions. Our results indicate that Spo0A is a likely target of inhibition. This could be direct or indirect. There could also be multiple targets, but the simplest model is currently that Spo0A is somehow …
Response to Reviewer #1 (Public Review):
Suggestions for improvement:
The authors show that the gene devI is necessary and sufficient for ICE-mediated delay of development initiation. Gene expression analyses suggest this delay affects the earliest stages of development (genes under control of spo0A, the master regulator of sporulation, are affected). I think the authors could investigate the mechanism of spo0A inhibition in more detail. Which aspect of spo0A function is affected by DevI? Starvation sensing, spo0A expression, activation of upstream kinases (KinA?), phosphorelay, or binding of Spo0A~P to promoters?
Excellent questions. Our results indicate that Spo0A is a likely target of inhibition. This could be direct or indirect. There could also be multiple targets, but the simplest model is currently that Spo0A is somehow affected. These are excellent questions to pursue in future work.
Ectopically expressed DevI (Fig 5) seemed to have a stronger inhibition of sporulation than ICEBs1 alone (Fig 2) - does the constitutively expressed protein block rather than delay sporulation? I wonder if the authors would like to comment on how, in the wild-type ICEBs1 context, DevI activity is eventually overcome by cells that eventually do sporulate after a delay. Furthermore, will cells that successfully transfer ICEBs1 be relieved of DevI-mediated sporulation inhibition?
Constitute expression of devI does indeed cause a stronger inhibition of sporulation than ICEBs1 alone. This is most likely because expression of devI in its normal context in ICE is likely transient. Expression of devI in ICE is regulated by the ICEBs1 cell-cell signaling genes rapI-phrI. In the wild-type context, accumulation of PhrI likely contributes to shutting off devI expression in time to allow sporulation. Lines 430-435 in the Discussion were modified slightly to make this more clear.
We do not expect that cells that successfully transfer ICEBs1 would be relieved of DevI-mediated sporulation inhibition, as a copy of the element is usually retained by the donor cell due to autonomous replication of ICEBs1 (and likely most or all ICEs) after excision from the host chromosome.
The data in Fig 4 suggest that devI is not the only ICEBs1-encoded factor providing a fitness advantage. Do the unknown factor(s) also delay development, or do they work via other mechanisms: i.e. does the ∆devI mutant have a sporulation delay? Any idea what the other factors might be (from bioinformatics for example)?
Excellent questions. We infer that the other (not devI) ICE-encoded factors that provide a fitness advantage are under control of the promoter Pxis (fitness is neutral in a Pxis deletion mutant). We do not yet know what this/these other factors are, nor do we know if they also inhibit development.
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Reviewer #3 (Public Review):
These authors report the identification of the function of a genetic determinant (dev1, formerly ydcO ) carried by the ICEBs1 element that increases fitness of the host strain by delaying the entry into the normal developmental pathway leading to biofilm formation and ultimately sporulation, such that the subpopulation expressing the product of dev1 increases in a mixed pool. An interesting novel aspect of the dev1 system is that it is co-regulated with ICEBs1 conjugation, and thus is only activated when the host strain is a minority of a mixed population; in this scenario the Dev1+ subpopulation is essentially cheating on the Dev-. Since expression of the Dev1 phenotype in an entire population would likely cause a crash, the ICE- population density-dependent regulation ensures that the fitness advantage …
Reviewer #3 (Public Review):
These authors report the identification of the function of a genetic determinant (dev1, formerly ydcO ) carried by the ICEBs1 element that increases fitness of the host strain by delaying the entry into the normal developmental pathway leading to biofilm formation and ultimately sporulation, such that the subpopulation expressing the product of dev1 increases in a mixed pool. An interesting novel aspect of the dev1 system is that it is co-regulated with ICEBs1 conjugation, and thus is only activated when the host strain is a minority of a mixed population; in this scenario the Dev1+ subpopulation is essentially cheating on the Dev-. Since expression of the Dev1 phenotype in an entire population would likely cause a crash, the ICE- population density-dependent regulation ensures that the fitness advantage disappears before the crash can occur. I think that the gene is interesting and this report adds a significant aspect to our understanding of the biology and evolution of ICE elements. Overall I am positive about this paper.
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Reviewer #2 (Public Review):
This manuscript provides convincing evidence that the ICEBs1 conjugative element confers a fitness advantage on the model bacterium B. subtilis during biofilm formation and sporulation. This effect is frequency dependent and is effected in large measure via an element gene, named devI, by an unknown mechanism that probably decreases the concentration of Spo0A-P. The data are well presented and successfully make the case for a fitness advantage conferred by the mobile element during biofilm formation and sporulation. It is likely that a mechanistic exploration of DevI will follow and will provide another facet to the regulation of Spo0A, a gift that keeps on giving.
Delaying sporulation in a mixed culture confers an advantage for the delayers. This has been convincingly shown. But I wonder about the effects …
Reviewer #2 (Public Review):
This manuscript provides convincing evidence that the ICEBs1 conjugative element confers a fitness advantage on the model bacterium B. subtilis during biofilm formation and sporulation. This effect is frequency dependent and is effected in large measure via an element gene, named devI, by an unknown mechanism that probably decreases the concentration of Spo0A-P. The data are well presented and successfully make the case for a fitness advantage conferred by the mobile element during biofilm formation and sporulation. It is likely that a mechanistic exploration of DevI will follow and will provide another facet to the regulation of Spo0A, a gift that keeps on giving.
Delaying sporulation in a mixed culture confers an advantage for the delayers. This has been convincingly shown. But I wonder about the effects in a clonal population of cells carrying ICEBs1 in competition with a null population. I appreciate that the delay in sporulation is transient, as pointed out in lines 404-407. But a delay of a few hours may be critical in this type of competition between populations as resources become limiting. This is presumably why sporulation is so exquisitely regulated on so many levels and in response to many external an internal signals. If so, ICEBs1 would have a deleterious effect and the element might be in danger of extinction. I suppose that an analogous discussion could be considered for biofilm formation.
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Reviewer #1 (Public Review):
Mobile genetic elements like phages, transposons, plasmids, and conjugative elements are widespread in prokaryotes and confer important traits to their hosts, including antibiotic resistance and virulence. In this study, the authors convincingly demonstrate that the mobile element ICEBs1 of Bacillus subtilis confers a fitness advantage to its host by delaying entry into metabolically costly developmental processes (biofilm formation and sporulation). The gene devI is identified as being responsible for delaying initiation of development, but the mechanistic basis for this could be further explored. Their results show that, in addition to conferring novel phenotypes, mobile elements exert influence by tuning existing host pathways, a paradigm that could be extended to many other prokaryotes.
Strengths:
The …
Reviewer #1 (Public Review):
Mobile genetic elements like phages, transposons, plasmids, and conjugative elements are widespread in prokaryotes and confer important traits to their hosts, including antibiotic resistance and virulence. In this study, the authors convincingly demonstrate that the mobile element ICEBs1 of Bacillus subtilis confers a fitness advantage to its host by delaying entry into metabolically costly developmental processes (biofilm formation and sporulation). The gene devI is identified as being responsible for delaying initiation of development, but the mechanistic basis for this could be further explored. Their results show that, in addition to conferring novel phenotypes, mobile elements exert influence by tuning existing host pathways, a paradigm that could be extended to many other prokaryotes.
Strengths:
The paper is written very clearly, the experimental data is convincing, the interpretations and conclusions are justified by the data.
The authors implemented clever genetic approaches to quantitatively compare the fitness of strains harboring or lacking ICEBs1 in co-culture. I appreciated the use of the conjugation mutant (comEK476E) to prevent ICE transfer that would confound the analysis. Similarly, the authors genetically separate the developmental pathways under which ICEBs1 confers an advantage (biofilm formation and sporulation), by deleting the spo0A promoter under sigH control to prevent sporulation but retain biofilm formation. Finally, to assess the contribution of ICE-encoded genes to fitness, the authors take advantage of a "locked-in" ICE variant (∆attR, oriT*) that cannot excise and replicate - thereby eliminating the confounding variable of gene dosage from ICE replication.
As mentioned above, the effects of ICEBs1 on development set an important precedent for how mobile genetic elements interact with their hosts. They are often regarded as autonomous elements, but the authors provide an example of how these elements can influence host pathways.
Suggestions for improvement:
The authors show that the gene devI is necessary and sufficient for ICE-mediated delay of development initiation. Gene expression analyses suggest this delay affects the earliest stages of development (genes under control of spo0A, the master regulator of sporulation, are affected). I think the authors could investigate the mechanism of spo0A inhibition in more detail. Which aspect of spo0A function is affected by DevI? Starvation sensing, spo0A expression, activation of upstream kinases (KinA?), phosphorelay, or binding of Spo0A~P to promoters?
Ectopically expressed DevI (Fig 5) seemed to have a stronger inhibition of sporulation than ICEBs1 alone (Figure 2) - does the constitutively expressed protein block rather than delay sporulation? I wonder if the authors would like to comment on how, in the wild-type ICEBs1 context, DevI activity is eventually overcome by cells that eventually do sporulate after a delay. Furthermore, will cells that successfully transfer ICEBs1 be relieved of DevI-mediated sporulation inhibition?
The data in Fig 4 suggest that devI is not the only ICEBs1-encoded factor providing a fitness advantage. Do the unknown factor(s) also delay development, or do they work via other mechanisms: i.e. does the ∆devI mutant have a sporulation delay? Any idea what the other factors might be (from bioinformatics for example)?
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Evaluation Summary:
All the reviewers were in agreement that this is an exceptionally rigorous paper that sets an important precedent for how mobile genetic elements can influence host biology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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