The hokW-sokW Locus Encodes a Type I Toxin–Antitoxin System That Facilitates the Release of Lysogenic Sp5 Phage in Enterohemorrhagic Escherichia coli O157

The toxin-antitoxin (TA) genetic modules control various bacterial events, such as plasmid maintenance, persister cell formation, and phage defense. They also exist in mobile genetic elements, including prophages; however, their physiological roles remain poorly understood. Here, we demonstrate that hokW-sokW, a putative TA locus encoded in Sakai prophage 5 (Sp5) in enterohemorrhagic Escherichia coli O157: H7 Sakai strain, functions as a type I TA system. Bacterial growth assays showed that the antitoxic activity of sokW RNA against HokW toxin partially requires an endoribonuclease, RNase III, and an RNA chaperone, Hfq. We also demonstrated that hokW-sokW assists Sp5-mediated lysis of E. coli cells when prophage induction is promoted by the DNA-damaging agent mitomycin C (MMC). We found that MMC treatment diminished sokW RNA and increased both the expression level and inner membrane localization of HokW in a RecA-dependent manner. Remarkably, the number of released Sp5 phages decreased by half in the absence of hokW-sokW. These results suggest that hokW-sokW plays a novel role as a TA system that facilitates the release of Sp5 phage progeny through E. coli lysis.


Introduction
The toxin-antitoxin (TA) system is a genetic module composed of a toxin and an antitoxin. Genes encoding these elements are generally contiguous and have been discovered in almost all sequenced bacterial genomes [1]. While a toxin arrests cell growth, the antitoxin neutralizes its toxicity [2]. TA systems have been implicated in a wide range of bacterial events, such as plasmid maintenance [3], persister cell formation [4][5][6], and phage defense [7][8][9][10].
The first TA locus was discovered in the E. coli plasmid in 1983 [11,12]. Since then, TA loci have been found not only in plasmids but also on bacterial chromosomes [13][14][15]. TA loci are also closely linked to prophage genomes. For example,~8% of the total TA loci have been overrepresented in the nine cryptic prophages of E. coli K-12 [16]. Cryptic prophages harbor mutations in genes required for virulence and phage formation, which render them trapped in the host genome. Although TA loci are also found in the genomes of functional prophages [17][18][19], the impact of their TA systems on the propagation of lysogenic phages remains poorly understood.
TA systems are currently classified into seven types according to the nature and function of antitoxins [20]. Type I antitoxins are small non-coding RNAs that bind to the cognate toxin mRNAs to inhibit translation or promote degradation [21,22]. The hoksok locus encodes a type I TA system found in various plasmids and bacterial genomes. Hok (host killing) is a hydrophobic peptide toxin that depolarizes the plasma membrane and causes cell growth retardation and lysis [23,24]. sok (suppression of killing) is a small non-coding RNA that inhibits translation of mok (modulation of killing) mRNA that overlaps with hok mRNA. The base-pair RNAs formed between sok RNA and mok mRNA are degraded by RNase III that specifically cuts double-stranded RNA [25]. Since translation of hok and mok mRNAs is coupled, sok RNA indirectly inhibits the translation of hok mRNA [3]. While this TA system present in the plasmid has been implicated in plasmid maintenance [26] and phage defense [27], the roles of hok-sok loci encoded in prophage genomes remain unclear.
In this study, we aimed to clarify the role of a hok-sok locus encoded in a prophage of the enterohemorrhagic E. coli O157: H7 Sakai strain. Bacterial growth and survival assays and phage plaque formation experiments have provided evidence that supports a new role of the hok-sok TA system which facilitates the release of Sp5 phage progeny through E. coli lysis.

hokW-sokW Functions as a TA System
E. coli O157: H7 Sakai strain has 18 prophages, three of which can infect nonpathogenic E. coli K-12 strains [28]. Among them, the lambdoid Sp5 (Sakai prophage 5) phage produces a major virulence factor called Shiga toxin 2. The genome of Sp5 phage encodes a gene pair named "hokW-sokW" that is predicted to be a member of the hok-sok family of type I TA system ( Figure 1A) [29]. HokW (51 amino acids) shares 44% identity and 69% similarity with Hok (52 amino acids) in E. coli R1 plasmid ( Figure 1B). We first examined whether hokW-sokW encoded in the Sp5 phage functions as a TA system. We constructed a plasmid pBAD24-hokW, in which a hokW is cloned under the control of an arabinose-inducible promoter. When HokW was expressed by arabinose in E. coli K-12 TY0807 cells, cell growth was immediately retarded ( Figure 1C). Resumption of cell growth around 4 h after arabinose addition is probably due to the depletion of arabinose. Next, we tested whether sokW RNA suppressed the toxic activity of HokW. We cloned hokW-sokW in an arabinose-inducible plasmid, pBAD18-hokW-sokW, in which hokW mRNA is expressed from an arabinose-inducible promoter and sokW RNA from its own promoter. When TY0807 cells harboring this plasmid were treated with arabinose, no obvious growth retardation was observed ( Figure 1D). To confirm the inhibitory effect of sokW RNA against HokW-mediated toxicity, we introduced mutations in the promoter sequence of sokW to repress its transcription ( Figure 1E; pBAD18-hokW-sokW(pro-less)). TY0807 cells harboring pBAD18-hokW-sokW(pro-less) exhibited growth retardation for approximately 3 h after arabinose addition ( Figure 1D). Importantly, the growth retardation observed in cells harboring pBAD18-hokW-sokW(pro-less) was partially restored by expressing sokW RNA using another plasmid, pACYC184-sokW ( Figure 1F). These results strongly suggest that sokW RNA counteracts the toxic activity of HokW. Taken together, we concluded that HokW is a toxin and that sokW RNA functions as an antitoxin against HokW.  Hok from E. coli R1 plasmid are shown. Red and blue letters indicate identical and similar residues, respectively. (C) TY0807 cells harboring pBAD24 or pBAD24-hokW were treated with or without L-arabinose (L-ara) when the OD660 reached 0.4 (indicated by the arrow) (D) TY0807 cells harboring pBAD18-hokW-sokW or pBAD18-hokW-sokW(pro-less) were treated with or without L-ara when the OD660 reached 0.4-0.5. (E) Nucleotide sequence around the -10 promoter element of sokW is shown. An asterisk is the transcription start site and arrows show nucleotides substituted in pBAD18-hokW-sokW(proless). (F) TY0807 cells harboring pBAD18-hokW-sokW(pro-less) plus either pACYC184-sokW or its vector pACYC184 were treated with or without L-ara when the OD660 reached 0.4.

hokW-sokW is a RNase III and Hfq Dependent Type I TA System
sokW RNA has a 71-nt complementary sequence to the 5′-UTR of hokW mRNA (Figure 1A). If sokW RNA forms a base pair in this region to repress hokW translation, it is likely that this process is RNase III-dependent [25]. To test this hypothesis, we performed cell growth assays with and without RNase III. While wild-type cells harboring pBAD18-hokW-sokW grew normally after arabinose addition, RNase III-deficient cells, ME5413, harboring the same plasmid exhibited growth retardation around 2 h after arabinose Hok from E. coli R1 plasmid are shown. Red and blue letters indicate identical and similar residues, respectively. (C) TY0807 cells harboring pBAD24 or pBAD24-hokW were treated with or without L-arabinose (L-ara) when the OD 660 reached 0.4 (indicated by the arrow) (D) TY0807 cells harboring pBAD18-hokW-sokW or pBAD18-hokW-sokW(pro-less) were treated with or without L-ara when the OD 660 reached 0.4-0.5. (E) Nucleotide sequence around the -10 promoter element of sokW is shown. An asterisk is the transcription start site and arrows show nucleotides substituted in pBAD18-hokW-sokW(pro-less). (F) TY0807 cells harboring pBAD18-hokW-sokW(pro-less) plus either pACYC184-sokW or its vector pACYC184 were treated with or without L-ara when the OD 660 reached 0.4.

hokW-sokW Is a RNase III and Hfq Dependent Type I TA System
sokW RNA has a 71-nt complementary sequence to the 5 -UTR of hokW mRNA ( Figure 1A). If sokW RNA forms a base pair in this region to repress hokW translation, it is likely that this process is RNase III-dependent [25]. To test this hypothesis, we performed cell growth assays with and without RNase III. While wild-type cells harboring pBAD18-hokW-sokW grew normally after arabinose addition, RNase III-deficient cells, ME5413, harboring the same plasmid exhibited growth retardation around 2 h after ara- binose addition (Figure 2A). This result suggests the involvement of RNase III. RNase III-mediated double-strand RNA degradation is likely to play a crucial role in the antitoxic activity of sokW RNA. We next examined whether the antitoxic activity of sokW RNA requires Hfq, an RNA chaperone that facilitates base-pairing between the two RNAs [30]. When HokW expression was induced in E. coli cells lacking Hfq, cell growth was partially retarded despite the presence of sokW ( Figure 2B). This result suggests that Hfq is involved in the antitoxic activity of sokW RNA and probably help base-pairing between sokW RNA and hokW mRNA. Together, these results suggest that hokW-sokW is a type I TA system whose antitoxin activity requires both RNase III and the Hfq chaperone.
Toxins 2021, 13, x FOR PEER REVIEW 4 of 14 addition ( Figure 2A). This result suggests the involvement of RNase III. RNase III-mediated double-strand RNA degradation is likely to play a crucial role in the antitoxic activity of sokW RNA. We next examined whether the antitoxic activity of sokW RNA requires Hfq, an RNA chaperone that facilitates base-pairing between the two RNAs [30]. When HokW expression was induced in E. coli cells lacking Hfq, cell growth was partially retarded despite the presence of sokW ( Figure 2B). This result suggests that Hfq is involved in the antitoxic activity of sokW RNA and probably help base-pairing between sokW RNA and hokW mRNA. Together, these results suggest that hokW-sokW is a type I TA system whose antitoxin activity requires both RNase III and the Hfq chaperone.

hokW-sokW Assists Growth Retardation and Reduces Cell Viability after Mitomycin C Treatment
hokW-sokW is encoded in the lysogenic Sp5 prophage of the E. coli O157: H7 Sakai strain. To explore the potential role of hokW-sokW in Sp5-mediated cell growth retardation, we compared cell growth and viability after Sp5 prophage induction with or without this type I TA system. For the experiment, E. coli K-12 MG1655 cells with Sp5 prophage were used. We first confirmed that deletion of hokW-sokW did not affect cell growth under normal conditions ( Figure 3A). The Sp5 prophage was induced in MG1655 cells using mitomycin C (MMC), a DNA-damaging agent that crosslinks two DNA bases and triggers prophage induction [31]. MMC is one of the most commonly used antibiotics for prophage induction. While MMC had no obvious effect on MG1655 cell growth, Sp5-containing cells (MG1655-Sp5) stopped growing around 2 h after MMC treatment and started to lyse around 3.5 h ( Figure 3B). Remarkably, growth retardation started approximately 40 min later in the absence of hokW-sokW (MG1655-Sp5(∆hokW-sokW)), indicating that this TA system facilitates Sp5-mediated growth retardation. To assess whether hokW-sokW also affects cell survival, we measured the number of viable cells at different time points after MMC treatment. At 30 min after the treatment, the cell viability of MG1655-Sp5 was comparable to that of MG1655-Sp5(∆hokW-sokW). However, the viability of MG1655-Sp5 dropped to about 55% of that of MG1655-Sp5(∆hokW-sokW) for the cells harvested 120 min ( Figure 3C). These results indicate that hokW-sokW not only facilitates growth retardation but also reduces cell survival.
HokW toxin is predicted to be a short peptide that destabilizes the plasma membrane. To examine whether hokW-sokW-mediated growth retardation is dependent on the lytic enzymes encoded in the Sp5 phage, we created a prophage that lacks both the R endolysin and the S holin (MG1655-Sp5(∆r-s)). The growth retardation of MG1655-Sp5(∆r-s) started approximately 3 h after MMC treatment, likely due to the production of Sp5 phages in the

hokW-sokW Assists Growth Retardation and Reduces Cell Viability after Mitomycin C Treatment
hokW-sokW is encoded in the lysogenic Sp5 prophage of the E. coli O157: H7 Sakai strain. To explore the potential role of hokW-sokW in Sp5-mediated cell growth retardation, we compared cell growth and viability after Sp5 prophage induction with or without this type I TA system. For the experiment, E. coli K-12 MG1655 cells with Sp5 prophage were used. We first confirmed that deletion of hokW-sokW did not affect cell growth under normal conditions ( Figure 3A). The Sp5 prophage was induced in MG1655 cells using mitomycin C (MMC), a DNA-damaging agent that crosslinks two DNA bases and triggers prophage induction [31]. MMC is one of the most commonly used antibiotics for prophage induction. While MMC had no obvious effect on MG1655 cell growth, Sp5-containing cells (MG1655-Sp5) stopped growing around 2 h after MMC treatment and started to lyse around 3.5 h ( Figure 3B). Remarkably, growth retardation started approximately 40 min later in the absence of hokW-sokW (MG1655-Sp5(∆hokW-sokW)), indicating that this TA system facilitates Sp5-mediated growth retardation. To assess whether hokW-sokW also affects cell survival, we measured the number of viable cells at different time points after MMC treatment. At 30 min after the treatment, the cell viability of MG1655-Sp5 was comparable to that of MG1655-Sp5(∆hokW-sokW). However, the viability of MG1655-Sp5 dropped to about 55% of that of MG1655-Sp5(∆hokW-sokW) for the cells harvested 120 min ( Figure 3C). These results indicate that hokW-sokW not only facilitates growth retardation but also reduces cell survival. Figure 3D). Notably, the retardation of cell growth was less severe in the absence o hokW-sokW (MG1655-Sp5(∆hokW-sokW ∆r-s)). These results strongly suggest that hokW sokW promotes growth retardation and possibly cell lysis independent of the lytic en zymes encoded in the Sp5 phage.

MMC Triggers HokW Toxin Production in a RecA Dependent Manner
These results suggest that MMC treatment induces HokW toxin expression. To ex amine this possibility, we first compared the amounts of hokW mRNA and sokW RNA before and after MMC treatment. As shown in Figure 4A, both hokW mRNA and sokW RNA were detected before MMC treatment, indicating that MMC does not promote hokW transcription. While hokW mRNA remained stable after MMC treatment, sokW RNA rap idly disappeared. These results suggest that HokW is produced after MMC treatmen Next, we measured the amount of HokW toxin using western blotting. Production of FLAG-tagged HokW (HokW-3FLAG) started 2 h after MMC treatment and plateaue HokW toxin is predicted to be a short peptide that destabilizes the plasma membrane. To examine whether hokW-sokW-mediated growth retardation is dependent on the lytic enzymes encoded in the Sp5 phage, we created a prophage that lacks both the R endolysin and the S holin (MG1655-Sp5(∆r-s)). The growth retardation of MG1655-Sp5(∆r-s) started approximately 3 h after MMC treatment, likely due to the production of Sp5 phages in the cells ( Figure 3D). Notably, the retardation of cell growth was less severe in the absence of hokW-sokW (MG1655-Sp5(∆hokW-sokW ∆r-s)). These results strongly suggest that hokW-sokW promotes growth retardation and possibly cell lysis independent of the lytic enzymes encoded in the Sp5 phage.

MMC Triggers HokW Toxin Production in a RecA Dependent Manner
These results suggest that MMC treatment induces HokW toxin expression. To examine this possibility, we first compared the amounts of hokW mRNA and sokW RNA before and after MMC treatment. As shown in Figure 4A, both hokW mRNA and sokW RNA were detected before MMC treatment, indicating that MMC does not promote hokW transcription. While hokW mRNA remained stable after MMC treatment, sokW RNA rapidly disappeared. These results suggest that HokW is produced after MMC treatment. Next, we measured the amount of HokW toxin using western blotting. Production of a FLAGtagged HokW (HokW-3FLAG) started 2 h after MMC treatment and plateaued around 3 h ( Figure 4B). Fractionated bacterial samples showed that HokW-3FLAG was enriched in the inner membrane, as seen with other members of the Hok family toxins ( Figure 4C) [23]. These results suggest that MMC treatment promotes HokW production and localization to the inner membrane through the disappearance of sokW RNA. around 3 h ( Figure 4B). Fractionated bacterial samples showed that HokW-3FLAG was enriched in the inner membrane, as seen with other members of the Hok family toxins ( Figure 4C) [23]. These results suggest that MMC treatment promotes HokW production and localization to the inner membrane through the disappearance of sokW RNA.
It is well known that treating lysogenic E. coli with a DNA-damaging agent such as MMC activates RecA, which stimulates the self-cleavage of the CI repressor to initiate prophage induction [32]. RecA is a single-stranded DNA-binding protein that is involved in DNA recombination and repair [33]. To examine whether HokW production triggered by MMC requires RecA, we constructed a deletion construct of recA in MG1655-Sp5(hokW-3FLAG) and measured the level of HokW ( Figure 4D). Western blotting revealed that HokW-3FLAG was not produced in ∆recA cells even 4 h after MMC treatment. This indicates that HokW production is RecA-dependent. HokW-3FLAG production was also observed when cells were shifted from 37 °C to 44 °C to trigger prophage induction ( Figure  4E). Notably, HokW-3FLAG was not produced when cells were treated with ampicillin that does not trigger prophage induction. These results strongly suggest that RecA-mediated prophage induction causes HokW production.
Finally, we examined the expression timing of HokW and the R endolysin after MMC treatment. Western blotting showed that HokW production started approximately 1 h earlier than the R (Figure 4B,F). This result suggests that HokW-mediated growth retardation and possibly cell lysis precedes cell lysis mediated by lytic enzymes, the R endolysin and the S holin, of Sp5 phage.  It is well known that treating lysogenic E. coli with a DNA-damaging agent such as MMC activates RecA, which stimulates the self-cleavage of the CI repressor to initiate prophage induction [32]. RecA is a single-stranded DNA-binding protein that is involved in DNA recombination and repair [33]. To examine whether HokW production triggered by MMC requires RecA, we constructed a deletion construct of recA in MG1655-Sp5(hokW-3FLAG) and measured the level of HokW ( Figure 4D). Western blotting revealed that HokW-3FLAG was not produced in ∆recA cells even 4 h after MMC treatment. This indicates that HokW production is RecA-dependent. HokW-3FLAG production was also observed when cells were shifted from 37 • C to 44 • C to trigger prophage induction ( Figure 4E). Notably, HokW-3FLAG was not produced when cells were treated with ampicillin that does not trigger prophage induction. These results strongly suggest that RecA-mediated prophage induction causes HokW production.
Finally, we examined the expression timing of HokW and the R endolysin after MMC treatment. Western blotting showed that HokW production started approximately 1 h earlier than the R (Figure 4B,F). This result suggests that HokW-mediated growth retardation and possibly cell lysis precedes cell lysis mediated by lytic enzymes, the R endolysin and the S holin, of Sp5 phage.

hokW-sokW Facilitates the Release of Sp5 Phage Progeny
Finally, we assessed whether the hokW-sokW TA system plays a role in phage propagation. We measured the number of Sp5 phage progeny released from MG1655 cells. Compared to wild-type, Sp5 phage progenies released from ∆hokW-sokW cells at 8 h after MMC treatment were reduced by around half (Figure 5). This suggests that hokW-sokW facilitates the release of Sp5 phage progeny.

hokW-sokW Facilitates the Release of Sp5 Phage Progeny
Finally, we assessed whether the hokW-sokW TA system plays a role in phage propagation. We measured the number of Sp5 phage progeny released from MG1655 cells. Compared to wild-type, Sp5 phage progenies released from ∆hokW-sokW cells at 8 h after MMC treatment were reduced by around half (Figure 5). This suggests that hokW-sokW facilitates the release of Sp5 phage progeny.

Discussion
TA loci are abundant in bacterial genomes and mobile genetic elements and are involved in various bacterial events, such as plasmid maintenance, persister cell formation, and phage defense. Here, we addressed the physiological role of a putative TA module encoded in the genome of a functional prophage in the E. coli O157: H7 Sakai strain.
We first demonstrated that hokW-sokW, encoded in the lysogenic Sp5 phage, functions as a type I TA system (Figures 1 and 2). When cells are exposed to extracellular stresses such as phage infection, type II TA genes are transcriptionally repressed, and consequently, labile antitoxins disappear and the toxins are activated [9,10,34]. In the hokW-sokW TA system, sokW RNA disappeared immediately after MMC treatment, and the remaining hokW mRNA was translated ( Figure 4A,B). Considering that MMC damages DNA, the disappearance of sokW RNA would be caused by blocking sokW transcription rather than augmenting mRNA degradation activity. MMC-mediated DNA damage results in the activation of RecA and initiation of prophage induction [31,32]. Intriguingly, HokW expression requires RecA ( Figure 4D). Thus, prophage induction rather than DNA damage itself is likely to trigger the production of HokW toxin. This idea is also supported by the result of Figure 4E. Prophage induction involves many biological processes, such

Discussion
TA loci are abundant in bacterial genomes and mobile genetic elements and are involved in various bacterial events, such as plasmid maintenance, persister cell formation, and phage defense. Here, we addressed the physiological role of a putative TA module encoded in the genome of a functional prophage in the E. coli O157: H7 Sakai strain.
We first demonstrated that hokW-sokW, encoded in the lysogenic Sp5 phage, functions as a type I TA system (Figures 1 and 2). When cells are exposed to extracellular stresses such as phage infection, type II TA genes are transcriptionally repressed, and consequently, labile antitoxins disappear and the toxins are activated [9,10,34]. In the hokW-sokW TA system, sokW RNA disappeared immediately after MMC treatment, and the remaining hokW mRNA was translated ( Figure 4A,B). Considering that MMC damages DNA, the disappearance of sokW RNA would be caused by blocking sokW transcription rather than augmenting mRNA degradation activity. MMC-mediated DNA damage results in the activation of RecA and initiation of prophage induction [31,32]. Intriguingly, HokW expression requires RecA ( Figure 4D). Thus, prophage induction rather than DNA damage itself is likely to trigger the production of HokW toxin. This idea is also supported by the result of Figure  4E. Prophage induction involves many biological processes, such as excision, cyclization, replication of phage DNA, expression of phage proteins, and assembly of phage particles. Further studies are required to clarify which of these processes lead to the loss of sokW RNA and HokW production.
Previous reports demonstrated that hok mRNA from E. coli R1 plasmid is slowly processed at its 3 -end after the transcription is stopped by rifampicin [3]. This truncated hok RNA is translated more actively than full-length hok mRNA [35]. In Figure 4A, two smaller RNAs than full-length hokW mRNA were detected and rapidly disappeared after MMC addition. Whether these RNAs correspond to the truncated hok RNA from R1 plasmid needs to be investigated. Although RNase III and Hfq were suggested to be required for sokW RNA-mediated translational repression of hokW mRNA, this antitoxic activity was partially effective in the RNase III and Hfq defective constructs ( Figure 2). Therefore, RNase III and Hfq are unlikely to play a major role in the sokW RNA-mediated translational repression. In line with this idea, neither RNase III nor Hfq mutants are lethal. Presumably, there is another mechanism for repressing HokW expression besides the cleavage of hokW mRNA by RNase III. And the base-pairing between hokW mRNA and sokW RNA would naturally occur in the absence of Hfq, albeit inefficiently.
The type I TA toxins are classified into two types: membrane-associated toxins and cytosolic toxins [36]. The membrane-associated toxins are small hydrophobic peptides that localize in the inner membrane, which affects the plasma membrane integrity and potential, and consequently leads to growth retardation and cell lysis [37,38]. Similarly, HokW are hydrophobic peptides ( Figure 1B) and are localized in the inner membrane ( Figure 4C). As expected, endogenous HokW production triggered by MMC facilitated growth retardation ( Figure 3B,D) and decreased cell viability ( Figure 3C). These results suggested that cell lysis would begin before the production of phage progeny is completed and that the number of phage progenies released would be decreased. However, contrary to this prediction, cells harboring hokW-sokW released phage progenies approximately 2-fold compared to the ∆hokW-sokW cells ( Figure 5). Based on these results, we propose a working hypothesis in which hokW-sokW plays a positive role in Sp5 phage propagation. If the Sp5 prophage does not encode the hokW-sokW TA locus, MMC treatment triggers the initiation of Sp5 prophage induction through RecA activation. Lysis enzymes are expressed at the late stage after induction, which results in cell lysis and the release of phage progeny ( Figure 6A). If the Sp5 prophage encodes hokW-sokW, MMC treatment triggers not only Sp5 prophage induction but also HokW production. HokW localizes to the inner membrane and partially causes cell lysis before lysis enzymes are fully expressed. Consequently, the release of Sp5 phage progeny starts earlier than usual ( Figure 6B).
The RnlA-RnlB TA system present in the cryptic CP4-57 prophage of the E. coli K-12 strain inhibits the propagation of lytic bacteriophage T4 [9]. Other TA systems (antiQ-AbiQ, ToxIN, MazE-MazF, etc.) have also been reported to function in defense against lytic phages [7,8,34,39]. Therefore, TA loci in the bacterial genome are believed to function as a mechanism that inhibits phage propagation. However, this study demonstrates that a TA system encoded in a lysogenic prophage has a positive effect on phage propagation, which is a new physiological role of TA systems. To date, the hok-sok TA system present in the R1 plasmid has been reported to inhibit T4 phage propagation [27]. In E. coli K-12 strain harboring a high-copy plasmid containing hok-sok, the efficiency of plating of T4 phage was reduced by 42% and plaque size was decreased by~85%. The molecular mechanism of phage inhibition by this hok-sok TA system remains unclear. Interestingly, TA systems belonging to the same family play opposite physiological roles in phage propagation depending on the type of mobile genetic element where the TA loci are located.