Phage-inducible chromosomal islands promote genetic variability by blocking phage reproduction and protecting transductants from phage lysis.

Phage-inducible chromosomal islands (PICIs) are a widespread family of highly mobile genetic elements that disseminate virulence and toxin genes among bacterial populations. Since their life cycle involves induction by helper phages, they are important players in phage evolution and ecology. PICIs can interfere with the lifecycle of their helper phages at different stages resulting frequently in reduced phage production after infection of a PICI-containing strain. Since phage defense systems have been recently shown to be beneficial for the acquisition of exogenous DNA via horizontal gene transfer, we hypothesized that PICIs could provide a similar benefit to their hosts and tested the impact of PICIs in recipient strains on host cell viability, phage propagation and transfer of genetic material. Here we report an important role for PICIs in bacterial evolution by promoting the survival of phage-mediated transductants of chromosomal or plasmid DNA. The presence of PICIs generates favorable conditions for population diversification and the inheritance of genetic material being transferred, such as antibiotic resistance and virulence genes. Our results show that by interfering with phage reproduction, PICIs can protect the bacterial population from phage attack, increasing the overall survival of the bacterial population as well as the transduced cells. Moreover, our results also demonstrate that PICIs reduce the frequency of lysogenization after temperate phage infection, creating a more genetically diverse bacterial population with increased bet-hedging opportunities to adapt to new niches. In summary, our results identify a new role for the PICIs and highlight them as important drivers of bacterial evolution.

Introduction

Bacteriophages (phages) are viruses that infect bacteria and are estimated to be the most abundant biological entities on the planet [1]. Phages play an important role in the ecology of biological ecosystems and affect not only bacterial population structures, but also global carbon and nitrogen turnover [2,3]. They are likewise key mediators of horizontal gene transfer (HGT) via transduction, one of the most important processes driving bacterial evolution as well as the spread of antimicrobial resistance (AMR) genes. Transducing particles that contain bacterial DNA are generated during phage infection or prophage induction by a variety of mechanisms that lead to three main types of transduction: generalized, specialized, and lateral transduction (recently reviewed in [4]). Generalized transduction is the process by which phages package any bacterial DNA (chromosomal or plasmid) and transfer it to another cell. Here, the phage terminase “mistakenly” recognizes pseudo-pac sites located on the host chromosomal or on episomal DNA and package it into the phage particles [5,6]. Conversely, specialized transduction can package and transfer a limited segment of host bacterial DNA adjacent to the phage [7]. This type of transduction is the result of the erroneous excision of a prophage from the host chromosome. Consequently, in addition to the phage DNA, a small segment of prophage-adjacent chromosomal DNA is also packaged into the phage capsid resulting in transducing particles containing, in most cases, a defective phage. Specialized transduction has been described in both cos-type and pac-type phages [8]. During lateral transduction, replication and packaging of the prophage genome begins before its excision [9-11]. Thus, the phage packaging machinery can continue into the host chromosome leading to the generation of transducing particle that contain larger segments of chromosomal DNA adjacent to the prophage integration site. Since packaging is directional, high frequency transducing particles are only generated for chromosomal DNA to one side of the prophage. This mechanism has only been observed in pac-type phages because of the additional packaging constraints imposed on cos-type phages render it far less likely to occur.

In the final stage of the lytic cycle, lysis of the host cells releases a mixture of phages and transducing particles, where phages significantly outnumber transducing particles. Once bacterial DNA is injected into a host cell, the transductants with the best chance of survival are either those with lysogenic protection or those that rapidly acquire immunity against the surrounding phages. The last few years have seen a vast expansion of known phage defense mechanisms and studies have revealed that some of these are clustered in what is called defense islands [12]. These mechanisms frequently act at the nucleic acid level (i.e. restriction modification systems or CRISPR-Cas) or trigger host cell suicide after infection (abortive infection (Abi) systems) (see [13] for a recent review). Defense systems that can allow the host cell to survive phage infection are of particular interest as they can promote the survival and expansion of transduced bacterial clones. For example, lysogenization (by transducing phages) and CRISPR-Cas systems have been shown to increase the frequency of host cells transduced with bacterial DNA by protecting the transductants from phage lysis [14,15].

Phage-inducible chromosomal islands (PICIs) are a widespread family of phage satellites that parasitize helper phages for their own benefit [4,16]. After helper phage infection or prophage induction of a PICI-containing strain, PICIs are activated by a phage-encoded inducer and excise from the bacterial chromosome, replicate extensively, and are then packaged into capsids comprised of phage-encoded proteins [17,18]. The Staphylococcus aureus pathogenicity islands (SaPIs) were the first PICIs to be described [19], and are the prototypical members of this family of mobile genetic elements (MGEs). Importantly, PICIs can interfere with phage reproduction by hijacking the phage packaging machinery to promote packaging of their own genomes at the expense of phage transfer or interfering with phage late gene transcriptional regulation [20-23]. For example, the interference mechanisms of SaPI1 lead to a reduction in phage titer and an increased survival of bacteria relative to a SaPI1-free strain infected with a phage [19]. Preliminary observations in this study also indicated that infected SaPI-containing cells were less likely to result in phage lysogens and that this could be either due to abortive or failed phage infection [19]. All PICIs to date carry at least some of these interference mechanisms although the extent of interference caused by individual PICIs on their inducing phage varies considerably. These interference mechanisms not only ensure high frequency SaPI transfer, but also enable the formation of transducing particles that use the PICIs strategy for packaging [24]. While many PICIs carry genes with obvious selective advantages such as virulence factors and antibiotic resistance [25-27], some PICIs do not, suggesting that these islands could provide unexpected advantages to their host cells to persist in nature.

Based on the known ability of SaPIs to interfere with phage replication, we hypothesized that SaPIs, like other phage defense mechanisms [15,28,29], might benefit the bacterial population by allowing for the survival of an increased number of transductants after horizontal acquisition of bacterial DNA. Here we report that SaPIs promote the survival of bacterial cells after phage-mediated gene transfer by interfering with phage reproduction and reducing the population of infective phage particles. Our results indicate that this protection occurs at the population level and that this is enough to protect most of the bacterial cells from subsequent rounds of phage attack, allowing both cells that have and have not received foreign bacterial DNA to survive and persist in the population. Our work identifies a hitherto unexplored function for SaPIs in generating genetic diversity and shaping bacterial populations and highlights the role of this family of MGEs in bacterial evolution.

Results

SaPIs increase transduction

SaPIs are the prototypical members of the PICI family [17], and were used here as a model to investigate the impact of PICIs on phage-mediated transduction. Specifically, three different SaPIs were selected: (i) SaPIbov1 (GenBank accession number AF217235.1) and SaPI1 (GenBank accession number U93688.2) are clinically important since they encode menstrual toxic shock syndrome toxin (TSST-1) and both have been extensively used for studying the mechanisms of PICI-phage induction, replication and phage interference; and (ii) SaPINY940, previously described as the incomplete phage pT1028, (GenBank accession number NC_007045.1) [30], which does not encode any identifiable toxins or virulence factors whose acquisition could be advantageous for its host. We further selected two temperate prophages (Φ11 and 80α) able to engage in generalized and lateral transduction to study the impact of each SaPI within a recipient strain on phage-mediated gene transfer.

To obtain transducing lysates, Φ11 or 80α lysogens carrying plasmid pJP2511 (CmR) or a chromosomal cadmium resistance cassette (CadR) next to the Φ11 or 80α attB sites were treated with mitomycin C (MC) for prophage induction [9]. Note that in these experiments the transfer of the plasmid (CmR) and the chromosomal (CadR) marker reflect generalized and lateral transduction, respectively [9,31]. Next, Φ11 lysates were used to infect (MOI 1:10) either the non-lysogenic, susceptible host strain, RN4220, or an RN4220 derivative carrying SaPIbov1, while 80α lysates were used to infect either RN4220 or RN4220 carrying either SaPINY940 or SaPI1 (see S1 Fig for an overview of the experimental setup). These combinations were chosen to generalize our results, and because Φ11 can induce SaPIbov1 [32], while 80α induces SaPINY940 and SaPI1. Note that SaPINY940 encodes a repressor identical to SaPI1, which is also induced by 80α [33]. Eighteen hours after infection, the different recipient cells were plated on selective media and the number of transductants were quantified. In support of our hypothesis that the presence of a SaPI in a recipient strain would be beneficial for the acquisition of foreign DNA by a bacterial population, the presence of either SaPIbov1, SaPI1 or SaPINY940 in the recipient host cells significantly increased the number of clones harboring the transduced CadR or CmR markers (Fig 1).

Fig 1

SaPIs promote the survival of transductants.

S. aureus RN4220 derivative strains either devoid of (red) or harboring either SaPIbov1 (blue), SaPINY940 (green) or SaPI1 (purple) were infected with an MOI of 1:10 (phage:bacteria) of the indicated phages and acquisition of a chromosomal cadmium resistance marker (lateral transduction) (top row panels) or a plasmid-borne chloramphenicol resistance cassette (generalized transduction) (bottom row panels) was determined 18 h post-infection. Bold horizontal lines in each boxplot represent the median and lower and upper hinges the first and third quartiles, respectively (n = 7 biological replicates for strains infected with either Φ11 or Φ11 Δdut or n = 3 biological replicates for strains infected with either 80α or 80α Δsri). Assessment of statistically significant differences between groups was performed using a two-sided Student’s t-test on log10 transformed data. p-values are indicated above the respective comparison, ns not significant. Limit of detection (LOD) for this assay is 10 transductants per ml.

Induction of the SaPI cycle is required to significantly increase transduction

In view of the previous results, we next analyzed whether the activation of the SaPI cycle in the recipient strain by the transducing phages was required to increase transduction. Interestingly, when we repeating the same experiments using Φ11 Δdut or 80α Δsri mutants, which do not encode the SaPIbov1 or SaPINY940/SaPI1 inducers, respectively [33], and therefore do not induce these SaPIs, the presence of either SaPIbov1 or SaPI1 did not increase transduction of the different markers (Fig 1). Unexpectedly, although the transduction frequencies observed for the 80α Δsri mutant-mediated transfer of the different markers into the SaPINY940-positive strain were significantly lower than those observed when the wt phage (able to induce SaPINY940) was used, we still observed a minor increase in the transfer of the markers into the SaPINY940-positive strain, compared to the transfer into the SaPINY940-negative strain (Fig 1). This result suggested that contrary to what is seen with SaPI1 or SaPIbov1, the presence of SaPINY940 can provide a residual increase in the transfer of the markers independently of the activation of the SaPI cycle. Nevertheless and in concordance with what was seen with the other islands, the transfer of the marker was much more efficient when the SaPINY940 cycle was induced. The molecular basis of the different behavior among different SaPIs is currently under investigation.

Next, since SaPI induction severely interferes with phage reproduction [17], we hypothesized that a reduction in the phage titer after the first few rounds of infection led to an increase in the number of cells that survived later rounds of phage attack (including the cells transduced with bacterial DNA). In support of this hypothesis, the titers of the wt Φ11 and 80α phages, but not of the Φ11 Δdut and 80α Δsri mutants, were severely reduced after infection of the strains carrying SaPIbov1 or SaPI1 (Fig 2A). This reduction in phage titers was linked to a significant increase in the number of survivors present in the bacterial population (Fig 2B). In accordance with the increased transduction observed when the SaPINY940-positive strain was infected with the 80α Δsri mutant, a reduction in phage titer (Fig 2A) and an increase in cell viability (Fig 2B) were observed after infection of the SaPINY940-containing strain by phage 80α Δsri.

Fig 2

SaPIs reduce phage reproduction and lysogenization but increase cell viability.

S. aureus RN4220 derivative strains either devoid of (red) or harboring either SaPIbov1 (blue), SaPINY940 (green) or SaPI1 (purple) were infected with an MOI of 1:10 (phage:bacteria) of the indicated phages. Phage titers (A), viable cells (B) and the relative number of lysogens per viable cell (C) for each recipient were determined. Note that the number of lysogens could not be determined for SaPINY940 due to an incompatibility of selection markers. Bold horizontal lines in each boxplot represent the median and lower and upper hinges the first and third quartiles, respectively (n = 7 biological replicates for strains infected with either Φ11 or Φ11 Δdut or n = 3 biological replicates for strains infected with either 80α or 80α Δsri). Assessment of statistically significant differences between groups was performed using a two-sided Student’s t-test on log10 transformed data. p-values are indicated above the respective comparison, ns not significant. Limits of detection (LOD) for this assay are 100 plaques per ml (A), 100 colonies per ml (B).

SaPIs do not increase HGT but protect transductants from phage attack

To address whether SaPIs promoted increased levels of HGT or promoted the survival of transduced bacterial populations, we followed cell viability, phage titers and transduction titers of marker genes after 1, 4 or 18 h post infection (Fig 3). Note that the one-hour time point post infection allows for the completion of the initial transduction event but does not provide enough time for the release of phage progeny from this first infection event. We observed no significant differences in either viable cell counts, phage or transduction titers at this time. This suggested that there was no inherent difference between the ability to acquire exogenous DNA via HGT in either SaPI-positive or SaPI-negative strains. However, differences began to be evident 4 h after the phage lysates were added to the recipients: SaPI-containing cells showed an increased number of viable cells and transductants as well as reduced phage titers relative to the SaPI-free recipient strains or strains infected with the phage inducer mutants (Fig 3). To estimate whether bacterial population underwent additional transduction events after the initial infection cycle, we calculated the ratio of transduced to viable cells at 1 h and 18 h post infection. In cases where subsequent transduction events occurred, this number should increase at the later timepoint, reflecting a larger portion of the bacterial population to have acquired the relevant resistance markers. We did not observe any increase in this ratio, further suggesting that transduction of the marker genes occurred during the first infection cycle and did not play a major role during later timepoints.

Fig 3

Assessment of strain viability, phage titers and transduced markers at different time points following infection.

The indicated strains either devoid of (red) or harboring either SaPIbov1 (blue) or SaPINY940 (green) were infected with an MOI of 1:10 (phage:bacteria) with the indicated phage donor lysates. Samples for viable cell count, phage titer and number of cells transduced with either antibiotic resistance marker were taken at the indicated timepoints. Note that at 0 h, viable cells correspond to the number of infected cells used, phage titers correspond to the number phages from the donor lysate added for the infection and resistance marker titers are 0 as the recipient cells are sensitive at this stage. Error bars are s.d. from the mean. Assessment of statistically significant differences between groups was performed using a two-sided Student’s t-test on log10 transformed data. p-values are as follows: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns not significant. Limits of detection (LOD) are 100 colonies per ml for viable cells counts, 100 plaques per ml for phage titers and 10 colonies per ml for transductions.

SaPIs protect bacterial populations through a range of MOIs

To test whether SaPIs could mediate population protection when exposed to higher initial phage burdens, we repeated the infection experiment using an MOI of 1:1 rather than the MOI of 1:10 that was used in the previous experiments (Fig 4). This high MOI was selected to ensure that most bacterial cells would undergo a phage infection (~63% at an MOI of 1:1 compared to ~9.5% at an MOI of 1:10 assuming Poisson distribution of infection) as well as to balance the drive of temperate bacteriophages to lysogenize at higher MOIs [34,35]. Even when cells were infected with 10-fold the number of phages, SaPI-positive cells retained an increased number of transductants, a reduced phage titer and increased viability compared to their SaPI-negative relative (Fig 4). These differences were more robustly maintained in experimental systems involving phage 80α compared to the system with Φ11, indicating that the afforded protection was likely dependent on the phage-SaPI pair as well as the infectious dose.

Fig 4

Impact of MOI on horizontal gene transfer, phage titers and viability.

S. aureus RN4220 derivative strains either devoid of (red) or harboring either SaPIbov1 (blue), SaPINY940 (green) or SaPI1 (purple) were infected with an MOI of either 1:1 or 1:10 (phage:bacteria) of the indicated phages and acquisition of a chromosomal cadmium resistance marker (lateral transduction) (A), a plasmid-borne chloramphenicol resistance cassette (generalized transduction) (B), phage titers (C) and cell viability (D) was determined 18 h post-infection. Bold horizontal lines in each boxplot represent the median and lower and upper hinges the first and third quartiles, respectively (n = 3–4 biological replicates as indicated). Assessment of statistically significant differences between groups was performed using a two-sided Student’s t-test on log10 transformed data. p-values are indicated above the respective comparison, ns not significant. Limit of detection (LOD) of transductants (A&B) is 10 transductants per ml, 100 plaques per ml for phage titers (C), and 100 colonies per ml for viable cells (D).

SaPIs increase genetic diversity in the recipient cells by reducing lysogenization

Prophages are known to promote transduction by protecting recipient cells from phage attack, preventing further superinfection by the same phage type (superinfection immunity) [36]. Consequently, most of the surviving transductants become lysogenic for the transducing phage in the absence of a PICI within the recipient population [14]. The fact that SaPIs modify phage dynamics prompted us to study the effect that SaPIs may have on the frequency of lysogeny after phage infection. To do that, we took advantage of the fact that the wt and mutant Φ11 and 80α prophages carried an erythromycin resistance marker ermC integrated into their phage genomes [37]. The lysates obtained in Fig 2A were used to infect either RN4220 (susceptible strain, Φ11 and 80α lysates) or RN4220 carrying either SaPIbov1 (Φ11 lysates) or SaPI1 (80α lysates), (MOI 1:10). Note that SaPINY940 was not used in these experiments because it carries the same ermC marker present in the phages. Eighteen hours after infection, the bacterial cells that survived the phage attack were grown on TSA plates lacking or containing erythromycin, and the proportion of the lysogens (compared to the viable cells) quantified. The SaPIbov1- and SaPI1-containing strains showed a significantly reduced proportion of cells becoming lysogenic compared to the susceptible RN4220 strain when infected with either wt Φ11 or wt 80α, respectively. By contrast, no differences between the SaPI-containing strains and RN4220 were observed when infecting them with either their respective inducer mutant lysates (Φ11 Δdut for SaPIbov1 and 80α Δdut for SaPI1) (Fig 2C).

The previous results suggested that the presence of the island will not only increase the survival of transductants but also the genetic diversity of the bacterial population by preserving part of the original non-lysogenic population and promoting the existence of additional different cell types, carrying different genetic information. To test this idea, we introduced into the lysogenic strains for Φ11 (wt or Δdut) both the chromosomally encoded Cd marker, and the plasmid pJP2511, which encodes CmR. These strains were MC induced, and the lysates were used to infect strain RN4220 or its SaPIbov1-containing derivative. From this experiment we determined the number of viable cells, lysogens, transductants as well as number of transductants becoming lysogenic by replica-plating them onto TSA supplemented with erythromycin. These data were then used to further assess the impact of SaPIs in a host cell on population diversity, by calculating the relative frequencies of different cell populations as a proportion of viable cells within each replicate. In support of the hypothesis, and as is shown in Fig 5, in absence of SaPIbov1, all the cells became lysogenic for Φ11 (Fig 5C, orange population, ErmR), which implied that the original non-lysogenic RN4220 strain (Fig 5C, grey population) was no longer present in the population. Moreover, in absence of SaPIbov1, two additional cell types appeared in the population, which corresponded to derivative lysogenic cells for Φ11 carrying either the chromosomally encoded CmR marker or the pJP2511 plasmid (Fig 5C, depicted in yellow). By contrast, the presence of SaPIbov1 notably increased diversity in the population, not only by modifying the frequency distribution of the aforementioned clones generated in absence of the island, but also by promoting the existence of three additional cell types (Fig 5). The first one corresponded to the original non-lysogenic RN4220 (grey population), which is absent when the island is not present but remained as the most prevalent clone in the population when the island is present. The other two clones were derivative RN4220 strains carrying the antibiotic resistance markers, but not the prophage (Fig 5, depicted in green). Of note is that the proportion of cells that had both become lysogenic for Φ11 and acquired one of the transduced resistance markers showed no differences between either the SaPI-positive or the susceptible RN4220 strain, suggesting that in the generation of this subpopulation (i) transduction and lysogenization were coupled, (ii) had to occur during early infection and (iii) were independent of the presence of a SaPI and other cell populations. Taken together, these results confirmed an unexpected role of the SaPIs in bacterial evolution by promoting genetic variability.

Fig 5

SaPIs increase population heterogeneity.

S. aureus RN4220 derivative strains either devoid of (RN4220) or harboring SaPIbov1 were infected with an MOI of 1:10 (phage:bacteria) of a Φ11 or Φ11 Δdut mutant lysate derived from a donor strains containing a chromosomal cadmium and plasmid-borne chloramphenicol resistance marker. Number of viable cells, transductants and lysogenization frequencies of the defined populations and their relative frequencies to the total number of viable cells were determined. Population frequencies of (A) cells acquiring the chromosomal cadmium resistance marker or (B) the plasmid-encoded chloramphenicol resistance marker in the defined recipient strains. Bold horizontal lines in each boxplot represent the median and lower and upper hinges the first and third quartiles, respectively (n = 4 biological replicates for all samples). Assessment of statistically significant differences between groups was performed using a two-sided Student’s t-test on log10 transformed data. p-values are indicated above the respective comparison, ns not significant. Note that 10−9 as relative population frequency in these data indicates populations that were completely absent. (C) Schematic representation of the presence of different cell populations in either a recipient lacking a SaPI (left) or containing a SaPI (right).

Discussion

Transduction is a fundamental process of bacterial evolution that hinges on the survival of the transduced cells (from further phage attack) in order to persist. By protecting their host cells from phage killing, phage defense mechanisms such as lysogenic protection or CRISPR-Cas, increase the chances of any given cell’s survival within the population while Abi-systems act on the whole bacterial population by preventing the release of phage progeny. Here we show that PICIs are not merely selfish MGEs but provide additional benefits to their host cells by blocking phage reproduction and thus promoting the survival of the entire bacterial population, including those that have acquired foreign genetic material (Fig 6). While prophages and CRISPR-Cas systems provide defense at the single cell level, PICIs are likely operating at population level through a general reduction in phage titers. While PICI-containing cells show no differences in phage infection, phage burst sizes are known to be reduced in PICI-containing cells that are unsuccessful in fully blocking the infecting phage [19]. Whether PICIs can also contribute to protecting individual cells from phage predation remains to be determined but is likely to be dependent on the individual phage-PICI combination. Untangling the protective effects that are eventually attributable to PICIs or simply the result of lysogenic protection (particularly at high MOIs), could provide further insights into the evolutionary impacts of these versatile molecular parasites. In this study, protection of host populations is dependent on the induction of the PICIs following host cell infection in all three PICIs tested. However, we cannot discard the possibility that some PICIs may confer protection even in the absence of phage induction. Indeed, some evidence for this can be gleaned from our results when infecting a strain containing SaPINY940 with a phage unable to induce this island. Even though a substantial amount of protection is lost in absence of island replication, there was still evidence of an increased level of protection in this strain background compared to its PICI-free relative. While this observation will require further confirmation, we anticipate PICI mediated protection might also act against non-inducing phages. This is currently under investigation.

Fig 6

Model of the impact of SaPI protection from phage predation on horizontal gene transfer and population heterogeneity.

In SaPI-free recipients (grey cells), infecting phages can reproduce without restrictions and lyse their host cell (indicated dashed lines and skull and crossbones symbol). Only lysogenization (cells shaded blue) can protect the host cell from future phage infection and killing. Any cells with horizontally acquired genes (red and green shaded cells) that do not undergo lysogenization are also subject to subsequent rounds of phage infection increasing the likelihood of their loss. In the presence of SaPIs, recipient strains can successfully block phage replication and can survive without the need for lysogenization. Consequently, transductants are also more likely to survive leading to an overall increase in population heterogeneity. We propose that this occurs mainly via a general reduction of phage reproduction and burst size protects at population level. However, SaPIs could also act at the individual cell level and protect the infected cell from lysis. The mechanistic details of protection are likely to be diverse and different SaPIs could employ replication-dependent and -independent mechanisms to achieve this.

Another interesting observation of our study is that SaPIs can substantially reduce the proportion of phage lysogens in the bacterial population which results in a more heterogenous population overall. Since lysogenization is a stochastic process that occurs randomly [38-40] and is promoted by high MOIs [34,35], we initially hypothesized that this reduction in the number of lysogens might simply reflect the reduction in phage titers caused by the SaPI interference. However, the consistent protection of the bacterial population and the resulting reduction in phage titers even at higher MOIs suggest that SaPIs might be more apt at blocking infecting phages than previously appreciated. In any case, such genetic heterogeneity can afford the bacteria with bet-hedging opportunities that enable them to better adapt and survive in changing and stressful environments.

While it is true that the horizontal transfer of specific genes can be beneficial to a cell if the acquired genes provide an advantage to the recipient cells compared to the rest of the population, it can also be detrimental if the mobilized genes have no function or are incompatible with existing genes [41]. The same applies to lysogenization, which can provide positive or negative effects, depending on the environmental context [42]. Importantly, our results demonstrate that SaPIbov1 increases the genetic variability of the recipient cells, not just by preserving the original lineage but also by creating new lineages that do not appear when the island is not present, highlighting how these events can further influence the evolution and clonal expansion of bacteria. Since the output of the transfer can be positive or negative for the recipient cells, PICIs can therefore increase the chance of the bacterial population to survive by minimizing the negative effects that the horizontal transfer of undesirable genes may have in the recipient cells by promoting genetic diversity. In essence, genetic variability is facilitated by the presence of PICIs in a host strain and natural selection will determine the fittest clone.

In nature, bacteria live in polymicrobial communities that contain abundant phage loads [43]. These phages are not only immensely important in shaping the bacterial community but are a major hub for the inter- and intrageneric exchange of genetic information [44]. Among the genetic material on offer are virulence and fitness factors as well as antimicrobial resistance genes. By increasing the frequency of genetic material exchange via increased survival of transduced clones, SaPIs can no longer be considered as self-serving MGEs but rather as major contributors to bacterial evolution. SaPIs benefit the host population by effectively acting as a host defense strategy, undermining phage reproduction, and increasing the chances of acquiring new sets of genes by transduction [45]. The fact that PICIs are found across distantly related taxa suggests that their lifestyle has a strong selective value representing a novel strategy for conferring fitness and promoting genomic variability among bacteria and adds another defense element to the mobile bacterial immune systems [29]. Following this rationale, the protective role of SaPIs, and other PICIs could potentially affect the application of phages as a means to reshaping microbiomes and eliminate specific pathogenic strains. This aspect should be evaluated in phage therapy and gene delivery to avoid the unintended fitness enhancement by transduction which could cause potential risks associated with public health and antimicrobial resistance.

Materials and methods

Bacterial strain and culture conditions

The bacterial strains and plasmids used in this study are detailed in Table 1. S. aureus strains were grown in Tryptic soy broth (TSB) or on Tryptic soy agar (TSA) plates. Antibiotic selection was used where appropriate (erythromycin 10 μg ml-1, chloramphenicol 20 μg ml-1, cadmium chloride 100 μM).

Table 1

Strains and plasmids used in this study.

Strain	Description	Reference
JP1996	RN4220 SaPIbov1 tst::tetM	[46]
JP4125	RN451 Φ11 Δdut	[33]
JP6022	RN10359 80α Δsri	[33]
JP6399	RN4420 lysogenic for 80α harboring an erythromycin resistance cassette	[47]
JP6400	RN4220 lysogenic for Φ11 harboring an erythromycin resistance cassette	[48]
JP14277	RN4220 SAOUHSC_01121::cadCA; cadmium resistance cassette inserted 35 kb downstream of Sa7 attB	[9]
JP19047	RN4220 harboring pT1028 marked with and erythromycin resistance cassette	[49]
JP19145	RN4220 SAOUHSC_01121::cadCA; cadmium resistance cassette inserted 5 kb downstream Sa5 attB	[9]
JP20714	JP14277 pJP2511	This study
JP20716	JP19145 pJP2511	“
JP20718	JP6022 with transduced with a lysate of JP6399 ermC (80α Δsri harboring an erythromycin resistance cassette)	“
JP20722	JP4125 transduced with a lysate of JP6400 resulting in Φ11 Δdut harboring an erythromycin resistance cassette	“
JP20844	JP20714 lysogenic for 80α harboring an erythromycin resistance cassette	“
JP20845	JP20714 80α Δsri harboring an erythromycin resistance cassette	“
JP20846	JP20716 lysogenic for Φ11 harboring an erythromycin resistance cassette	“
JP20847	JP20716 lysogenic for Φ11 Δdut harboring an erythromycin resistance cassette	“
RN10359	RN450 lysogenic for 80α	[50]
RN10616	RN4220 lysogenic for 80α	[50]
RN10822	RN4220 SaPI1 tst::tetM	[51]
RN450	NCTC8325 cured of Φ11, Φ12 and Φ13	[52]
RN451	RN4510 lysogenic for Φ11	[50,52]
RN4220	Restriction-defective derivate of RN450	[53]
Plasmids	Description	Reference
pJP2511	Gram-positive plasmid containing a chloramphenicol resistance cassette, CmR	[54]

Phage induction and titration

S. aureus strains lysogenic for the defined phages and containing the defined chromosomal markers or plasmids were grown to early exponential phase (OD540~0.15) at 37°C and 120 rpm. Cultures were then induced by the addition of mitomycin C (2 μg ml-1) and incubated for 4–5 h at 30°C followed by overnight incubation at room temperature before filtering with a 0.2 μm syringe filter (Sartorius). To determine the phage titers, RN4220 cultures were grown to OD540~0.35 and 100 μl of this culture was mixed with 3 ml of phage top agar (PTA, 20 g l-1 Nutrient Broth No. 2, Oxoid, plus 3.5 g l-1 agar, Formedium supplemented with 10 mM CaCl2) and overlaid onto phage base agar plates (20 g l-1 Nutrient Broth No. 2, Oxoid, plus 7 g l-1 agar, Formedium supplemented with 10 mM CaCl2). Phage lysates and dilutions in phage buffer (PHB) (1 mM MgSO4, 4 mM CaCl2, 50 mM Tris-Cl, 100 mM NaCl, pH = 8) were spotted in triplicates of 10 μl each onto lawns of the specified strains, dried and incubated overnight prior to plaque forming unit (PFU ml-1) determination.

Phage time-course infection experiment

Cultures of the indicated strains were grown to exponential phase and normalized to an OD540 of 0.25 corresponding to ~1 x 108 CFU ml1. Five to ten ml of this suspension were then supplemented with 5 μM CaCl2 and infected with the defined phage lysates at an MOI of either 1:10 (phage:bacteria) and incubated at 30°C and 80 rpm. At the indicated timepoints, samples were taken to assess the number of viable cells, transductants and phage titers.

Viable cell count determination

Cultures were serially diluted in PBS and spotted in triplicates of 10 μl per dilution onto a TSA plate. The plates were incubated for 18 h and the colony forming units determined.

Transductions of resistance markers

At the defined timepoints, 100 μl of culture was plated directly onto TSA supplemented with the appropriate antibiotics. Plates were then incubated for 24–48 h (depending on the resistance marker assessed) and colonies were counted for transduction titer enumeration (TFU ml1).

Phage lysogenization

Lysogenization of Φ11 was assessed either directly from culture or by replica plating surviving colonies or transductants onto TSA plates supplemented with 10 μg ml-1 erythromycin, as this phage contained an erythromycin resistance cassette within its genome.

Statistical analysis

Statistical analysis and plotting was performed using RStudio version 1.4.1717 and R version 4.1.0 [55] with the following packages installed: ggplot2 [56], tidyverse [57], stringi [58], plyr [59], dplyr [60], rstatix [61] and datarium [62]. Details are annotated in the relevant figure legends.

Experimental set up.

Recipient cultures were grown to exponential phase (OD540 of 0.25, corresponding to ~1 x 108 CFU ml1) in TSB. 5 ml of each recipient culture supplemented with 5 μM CaCl2 were infected with indicated phage lysates at an MOI of 1:10 (phage:bacteria). Cultures were incubated at 30°C and 80 rpm and samplings were performed at the indicated timepoints (1, 4 and 18 h). For transduction assessment, 100 μl of each culture at the defined timepoints were plated directly onto TSA plates supplemented with the appropriate antibiotics. For viability assessment, cultures were serially diluted in PBS and spotted in triplicates of 10 μl per dilution onto TSA plates. For phage titer assessment, 1 ml of each culture was filtered and used for serial dilutions in phage buffer (PHB). Dilutions were spotted in triplicates of 10 μl each onto PBA plates overlaid with a lawn of RN4220. Created with BioRender.com.

(TIF)

Click here for additional data file.

22 Jun 2021

Dear Jose,

Thank you very much for submitting your Research Article entitled 'Phage-inducible chromosomal islands promote genetic variability by blocking phage reproduction' to PLOS Genetics.

As you will see below, the reviewers' opinions on your manuscript are diverse. I invite you to revise your manuscript paying close attention to the reviewers' comments, especially considering the relevant objections raised by reviewers #1 and #3.

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

If you decide to revise the manuscript for further consideration at PLOS Genetics, please aim to resubmit within the next 60 days, unless it will take extra time to address the concerns of the reviewers, in which case we would appreciate an expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments are included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool.  PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

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[LINK]

We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.

Yours sincerely,

Josep Casadesús

Section Editor: Prokaryotic Genetics

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Ibarra-Chavez et. al. present measuring the impact of phage-induced SaPI activity on rates of resistance marker acquisition in bacterial recipient cells. They observe increased transduction of resistance markers and decreased lysogeny of inducing phages when bacterial hosts encode SaPIs. From this, the authors conclude that SaPIs actively promote host DNA co-transduction to increase host population genetic diversity and thus increase survival. The work is presented in a concise manner and I appreciated the exploration of the potential importance a non-toxigenic SaPI. Unfortunately, I disagree with the conclusions the authors draw from the data. I find the work to be lacking in novelty and consideration of several major factors that are invalidating to the conclusions in the text (see major points below). I do not recommend publication of this work in any journal without serious reconsideration of the scope of the paper.

Major points

1. The manuscript (including the statement in lines 78-79) fails to acknowledge what is currently already known in the broad field of PICI biology regarding PICI-mediated interference with phages. Being familiar with the literature, it is unclear what new biological insights are obtained from this work.

2. Multiple figures in the text do not enrich current understanding of the SaPI system but rather reiterate known information with the tone of novelty.

a. Figure 2 shows that phage titer is decreased with SaPI activity. This phage repression activity has previously been explored in molecular detail (Ram et. al. PNAS 2014). The reduction in phage titer is an expected result from what is already known about the system

b. Figure 2 also shows increased cell viability in the presence of SaPIs, which was demonstrated in the original molecular characterization of the system (Ruzin et. al. Mol Micro 2001) and continues to be a theme in SaPI biology. Similarly, Figure 3 shows a decrease in lysogenization frequency in hosts with active SaPIs, another phenomenon described in the original molecular characterization of SaPIs by Ruzin et. al.

3. The data and resulting analysis confuse rather than clarify a fairly intuitive concept: if a host cell harbors a phage-interference mechanism like a SaPI, phage infection will be impaired resulting in decreased lysogenization (because fewer phage particles can be produced) and increased host population survival which translates to increased probability of bacterial DNA uptake by transduction. I would argue that any mechanism that impairs phage reproduction in a population could effectively result in the same phenomenon. Thus, there does not appear to be any new biological insights gleaned from this work.

4. The authors draw the conclusion that increased population diversity of recipient cells containing SaPIs acts as an ecological bet-hedging strategy to promote survival in stressful conditions. I do not believe that the data presented in this manuscript directly support this claim. Further assessment of the relative fitness of these populations is required in order to make such a claim about an ecological strategy.

Minor points

1. (Lines 44-48) It is unclear whether all surviving cells in these populations have acquired new DNA.

2. (Lines 54-55) The different types of transduction (generalized vs. specialized vs. lateral) need some clarifying definition in the text.

3. Line 87 the designation of the SaPI pT1028 is unclear; this nomenclature is appropriate for a plasmid. If the original publication (is there a citation for this SaPI?) erroneously referred to this element as a plasmid it should be corrected and not perpetuated in the literature

4. Lines 89-90 could be clarified by stating here (rather than later) that these phages are the inducing phages for the SaPIs. I would also like some clarification on cross-activity between the selected SaPI-phage pairs (i.e. can phi11 induce pT1028 and 80alpha induce SaPIbov1?)

5. A model of the experimental set-up would greatly aid the reader’s understanding of lines 80-101.

6. Consider referring to RN4220 and other strain numbers as something more understandable to the general reader (e.g. “susceptible host”)

7. Please refer to the specific sub-panel of figures referenced in the text (e.g. line 128 should specifically reference Figure 3A)

8. Line 77 wording requires clarification regarding PICI and host DNA co-transduction.

9. Line 130 and elsewhere, please detail the actual degree of reduction rather than just calling it statistically significant.

10. Lines 175-181 need editing, there are words missing from some of these sentences that are required for grammatical correctness and clarity, specifically “creating news that” and “since as mentioned the output”

11. Figures 1-4: please provide information about the limit of detection for these assays.

12. Figures 1, 2, 3B: One-way ANOVA statistical analysis is not appropriate for these data.

13. Figures 1B, 2B: It appears that there is still some SaPI activation in the absence of sri in 80alpha. This is not addressed in the text.

14. Figure 3: Experiments conducted in duplicate are compared to experiments conducted 5 times. This seems inappropriate considering nearly all other experiments were conducted in biological triplicate at minimum.

15. Figure 4: Individual data points on the bar graphs (as in Figures 1-3) are absent on this figure. This figure would also be strengthened by inclusion of a third biological replicate.

16. Figure 5 is extremely confusing and has many elements that are not factually correct. It could be clarified by removing the shading in the large squares, including the initial phage infection step to orient readers to the overall process, and some serious reconsidering of arrow colors. This figure also contains some misleading information, specifically in the right-side panel that shows an individual SaPI+ cell surviving phage infection (it does not in fact protect the individual cell) and successful transduction blocking further phage infection (also not supported by what is currently known). This figure should be seriously reconsidered.

Reviewer #2: Interesting manuscript pointing to a central role of PICI's in transduction and importantly also for lysogenization which could be highlighted more and discussed: Why do bacteria avoid becoming lysogens and rather carry PICIs?

Line 32: A more diverse - more than what? The statement of diversity is somewhat overstating the results of the manuscript. Basically, the diversity is obtained by cells being able to survive without being lysogens - not that a greater diversity of cells in general or transduced cells are being created. The abstract should put more focus on the fact that more cells are able to survive in a phage environment without becoming lysogens.

line 94: Why is RN4220 being used here? The strain seems in general to suffer from several problems so a strain not having undergone rounds of mutagenesis would be preferable

Figure 2, line 368: Are the viable cells transductants or just viable cells in culture?

Figure 3 and figure 4: You are not commenting on the results of figure 3B in the text and basically the data in figures 3 and 4 seem in part to be the same but presented in different ways.

line 177: new ones instead of news?

Reviewer #3: The manuscript by Ibarra-Chávez et al, entitled "Phage-inducible chromosomal islands promote genetic variability by blocking phage reproduction" describes a new role for PICIs as promoters of phage-driven gene transfer. The data provided for two different PICIs, induced in the presence of two different phages in Staphylococcus aureus are convincing and overall support the model provided in Fig. 5. The main caveat of the manuscript is that it requires extensive prior knowledge of the PICIs biology and bibliography. The introduction is rather minimalist and does not provide sufficient information to guide the reader toward the initial hypothesis. This version needs improvement to reach the large audience and scope of the journal.

L.62: The description and biology of PICIs is rather minimalist, and not really adapted to a large audience. In particular, the phage-encoded inducer needs additional description as it is needed to understand Fig. 1.

L. 75, What is meant by “local population?”, if the model considers mixed or non-homogenous populations, it could be further explained.

L.79, it is not clear from the abstract or introduction how the work provided here relates to bacterial (genome) evolution.

L. 84-88: Two different SAPIs and cognate phages were used in this study for the sake of generalization (as indicated l. 96). Although the use of SapIbov1 is clearly explained, it is hard to get why pT1028 was chosen? A more detailed justification and the addition of references for these two SAPIs models would help the reader. In addition, as the authors study many different PICIs models, why not including different strains and classes of PICIs from different bacterial strains in the study to generalize even more?

L.93: Generalized and lateral transduction should be defined in the introduction to understand the subtility of using 2 different reporters.

L. 94: Strain RN4220 is a restriction-defective derivative of RN450, as indicated in Table 1. Why such a strain is needed. Table 1 could also mention the genotype of the original RN450.

L.97, is there a reference to provide for pT1028 induction by 80α?

L.99, the hypothesis mentioned could be rephrased to help the reader.

L. 110, it is hard to understand the basis of such hypothesis without additional explanation. If the phage titer decreases, is it because each cell is less productive, or because cells become less susceptible?

L. 117, the link between lysogeny and transduction facilitation is not obvious as stated.

L. 156, in this experiment the genetic variability observed is driven by the co-occurrence, and maintenance in the overall population, of several type of cells. Does it influence the population heterogenicity on the long term? How were the time of plating (18 h post-infection) chosen? In other words, is it possible to obtain a different distribution depending on the incubation time?

Discussion: Could the authors precise why they mention lysogenisation is a stochastic process and provide the appropriate references?

As stated in the text, the heterogenicity of the population driven by SAPIs could provide an advantage to the overall population. This could be tested experimentally.

Additional minor points: Phage titer instead of “phage titre”, several occurrences. Strain RN450 is missing in Table 1.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: No: Figure 4 represents relative population frequency and the actual counted number of cells are not provided

Reviewer #3: Yes

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

29 Aug 2021

Submitted filename: Response to reviewers.docx

Click here for additional data file.

21 Sep 2021

Dear Jose,

Thank you very much for submitting your Research Article entitled 'Phage-inducible chromosomal islands promote genetic variability by blocking phage reproduction' to PLOS Genetics.

As you will see below, reviewer #1 expresses disappointment about the fact that major concerns cited in the first round of review have not been addressed. I must agree with their view. Reviewer #3 also expresses a similar opinion in comments to the editor (but their comments to the authors are certainly more positive). After pondering the situation, I think that the right decision is to give you a second opportunity to address the comments made by reviewer #1. Please note that dismissal of criticism should not be customary.

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

If you decide to revise the manuscript for further consideration at PLOS Genetics, please aim to resubmit within the next 60 days, unless it will take extra time to address the concerns of the reviewers, in which case we would appreciate an expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments are included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool.  PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, use the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.

Yours sincerely,

Josep Casadesús

Section Editor: Prokaryotic Genetics

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The manuscript re-submitted by Ibarra-Chavez et. al. includes positive revisions increasing the amount of background information in the introduction and substantial figure revisions that have made the data more aesthetically pleasing. However, the authors failed to address the major concerns cited in the first round of review. Rather than constructively responding to the critiques raised, the authors chose to dismiss nearly every major critique I provided with little to no attempt to address the logic or concern itself. The authors instead chose to re-state their original hypotheses and decline to include any additional experiments or controls that would strengthen the claims they are trying to make. The manuscript continues to fail to acknowledge what is currently already known in other PICI systems where a role in protecting bacteria from phage predation has already been detailed. It is not to say that there are not additional experiments to be done in this area, there clearly are, however if a reader new to this field picked up this paper, they would assume based on how it is written that PICIs interfering with phages has been shown only in this system and here for the first time. Rather than continue to provide detailed feedback on this manuscript I will simply re-iterate a few crucial details that are particularly troubling:

1. The model in figure 5 has undergone edits and is still extremely misleading and unclear. The authors conduct experiments at MOI 1:10 (phage:bacteria) and do not see a change in cell viability in SaPI+ hosts, which they have interpreted here as SaPI+ cells surviving phage infection at the single-cell level. This data simply cannot be interpreted as survival or SaPI-mediated protection at the single-cell level. At the very least a high MOI infection should be performed to test this hypothesis directly and provide actual supporting data. It is also extremely unclear from the model that transduction results from an initial round of phage infection.

2. The SaPINY940 data is not explained satisfactorily in the text. SaPINY940 is stated to be induced by sri but the citation for this information (Ref. 26 Tormo-Mas et. al. Nature 2010) does not include this SaPI in its analysis. It is unclear how the authors conclude that sri is the inducer and unclear if there is SaPI induction in the absence of sri. From the data in this manuscript, it appears there is some level of induction without the inducer, which makes this SaPI a questionable choice to include in this analysis attempting to connect co-transduction with SaPI activation. If the authors wish to suggest that SaPINY940 has some additional phage resistance capacity outside direct phage parasitism, this should be explored more thoroughly, especially considering the conceptual similarity to recent discoveries in other satellite-encoded defense systems (e.g. Bikard Lab https://www.biorxiv.org/content/10.1101/2021.01.21.427644v1). I expect the authors to conclude such investigation is outside of the scope of the current manuscript, I can agree with that provided that the net result of such protection (at the single cell level) is not included in the model (figure 5).

3. I do not agree that the authors have discovered a novel role for SaPIs (or PICI’s in general) in diversity-generating transduction. The authors do not include controls to convince readers that SaPIs specifically (and not any phage defense in general) can have this effect.

4. I disagreed with the author’s conclusion that SaPIs promote host DNA co-transduction to increase host population genetic diversity. The authors responded “our main conclusion is not that SaPIs are actively promoting host DNA co-transduction but rather that SaPIs promote the survival of transductants by protecting the overall population and potentially individual cells from phage predation”. If that is the case the authors should more carefully state their claims, for example, the authors have chosen to title Figure 1 “SaPIs promote increased levels of horizontal DNA transfer.” These details are in direct conflict with the statement in the authors’ rebuttal.

5. A note about statistics: I am not questioning general significance of the differences being claimed, the averages are obviously different by eye. However, ANOVA analysis with post hoc EMM is not the most appropriate method for analyzing these data sets. Every comparison made in this manuscript is between 2 well-defined groups that do not appear to have similar distributions. This type of data is best analyzed by Student t-test, which does not assume that your data are distributed the same in all sets. ANOVA assumes all sets have equal distribution and attempts to find relevant pairs of groups to analyze. The experimental set-up here defines these groups so this is not necessary. A single t-test per group results in the lowest probability of making a Type I error. Choosing ANOVA and then post hoc analysis may “work”, but it increases the risk of Type 1 error, artificially inflates the p-value, and is an unnecessarily complicated analysis for the type of data being presented in this manuscript.

Reviewer #2: I am happy with the response

Reviewer #3: Overall, the text of the manuscript has improved.

Title could be completed: Phage-inducible chromosomal islands promote genetic variability by blocking

phage reproduction and protecting the transductants from phage lysis

Line 41: “of the general bacterial population”, this is not obvious what is meant by general population, at the population level ?

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: None

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

9 Feb 2022

Submitted filename: 20220208 Response to reviewers.docx

Click here for additional data file.

1 Mar 2022

Dear Jose,

Thank you very much for submitting a revised version of your manuscript 'Phage-inducible chromosomal islands promote genetic variability by blocking phage reproduction and protecting transductants from phage lysis' to PLOS Genetics. As you will see below, the manuscript has been examined by one reviewer (chosen from those who had evaluated previous versions). Based on the reviewer's comments, I think that an additional round of review might improve the story. Therefore, I invite you to consider the reviewer's objections. Then you may either modify the manuscript or explain why you disagree with the reviewer if such is the case.

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We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

Please let me know if you have any questions while making these revisions.

Yours sincerely,

Josep Casadesús

Section Editor: Prokaryotic Genetics

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The manuscript is much improved from the initial submission, but there are some points on which it appears we will have to agree to disagree. The experiments that were used to conclude that "SaPIs protect individual cells from phage attack" are inappropriate and do not make any sense to me. At an MOI=1, ~63% of cells are expected to be infected, unless the authors have a compelling reason to indicate the Poisson distribution does not apply to the system under study.

The authors state: "We reasoned that if SaPI-mediated protection acted at a population level alone, we should not observe any differences in cell viability, phage, or transduction titers between SaPI-positive and SaPI-negative recipient cells under these conditions. "

I disagree. Seeing as nearly 40% of the cells are uninfected, and phage titers are reduced following an initial round of infection by the SaPI it is no wonder the authors have a difference in cell viability. This does not support the model that SaPIs protect individual cells from phage attack. At an MOI =5, 99% of cells would be infected and I am certain that the authors would find absolutely no difference in cell survival +/- SaPI, but that is not the experiment they chose to perform.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

12 Mar 2022

Submitted filename: Response to reviewers.docx

Click here for additional data file.

14 Mar 2022

Dear Jose,

I am pleased to inform you that your manuscript entitled "Phage-inducible chromosomal islands promote genetic variability by blocking phage reproduction and protecting transductants from phage lysis" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

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In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

If you have a press-related query, or would like to know about making your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Josep Casadesús

Section Editor: Prokaryotic Genetics

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

----------------------------------------------------

Comments from the reviewers (if applicable):

----------------------------------------------------

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25 Mar 2022

PGENETICS-D-21-00720R3

Phage-inducible chromosomal islands promote genetic variability by blocking phage reproduction and protecting transductants from phage lysis

Dear Dr Penadés,

We are pleased to inform you that your manuscript entitled "Phage-inducible chromosomal islands promote genetic variability by blocking phage reproduction and protecting transductants from phage lysis" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript.

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Thank you again for supporting PLOS Genetics and open-access publishing. We are looking forward to publishing your work!

With kind regards,

Zsofia Freund

PLOS Genetics

On behalf of:

The PLOS Genetics Team

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