Staphylococcal interference with helper phage reproduction is a paradigm of molecular parasitism

Geeta Rama, John Chena, Krishan Kumara, Hope F. Rossa, Carles Ubedaa,1, Priyadarshan K. Damleb, Kristin D. Laneb, José R. Penadésc, Gail E. Christieb, and Richard P. Novicka,2

aSkirball Institute Program in Molecular Pathogenesis and Departments of Microbiology and Medicine, New York University Medical Center, New York, NY 10016; bDepartment of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298-0678; and cDepartment of Genomics and Proteomics, Instituto de Biomedicina de Valencia (IBV-CSIC), 46010 Valencia, Spain

Edited* by Sankar Adhya, National Institutes of Health, National Cancer Institute, Bethesda, MD, and approved August 3, 2012 (received for review March 19, 2012)

Staphylococcal pathogenicity islands (SaPIs) carry superantigen burst size was greatly reduced, and survival of phage-infected cells and resistance genes and are extremely widespread in Staphylo- was increased (11). It was observed at that time that SaPI1 was coccus aureus and in other Gram-positive bacteria. SaPIs represent packaged in small infectious particles, and it was soon shown that a major source of intrageneric and a many SaPIs contain two or more capsid morphogenesis (cpm) stealth conduit for intergeneric gene transfer; they are phage sat- genes (2, 12, 13) responsible for diverting phage virion proteins ellites that exploit the life cycle of their temperate helper phages to small capsid formation. It was hypothesized that the diversion with elegant precision to enable their rapid replication and pro- of phage virion proteins to the formation of these particles was responsible for interference (8). miscuous spread. SaPIs also interfere with helper phage reproduc- fi tion, blocking plaque formation, sharply reducing burst size and In this report, we con rm this hypothesis and go beyond it to demonstrate that there are other mechanisms of phage in- enhancing the survival of host cells following phage infection. Here, terference. Some SaPIs use one mechanism, some use a second, we show that SaPIs use several different strategies for phage in- some use both, and some add a third. We suggest that inter- terference, presumably the result of convergent evolution. One ference is not simply a consequence of competition between MICROBIOLOGY strategy, not described previously in the microcosm, SaPI and phage for “goods and services,” but is a key feature of fi involves a SaPI-encoded protein that directly and speci cally inter- the SaPI lifestyle. feres with phage DNA packaging by blocking the phage terminase small subunit. Another strategy involves interference with phage Results reproduction by diversion of the vast majority of virion proteins To begin these studies, we questioned whether the SaPI1 inter- to the formation of SaPI-specific small infectious particles. Several ference mechanism was universal, and immediately realized that SaPIs use both of these strategies, and at least one uses neither it could not be, because several of the known SaPIs, including but possesses a third. Our studies illuminate a key feature of the SaPIbov2 (1) and SaPIbov5 (see below), do not contain homo- evolutionary strategy of these , in addi- logs of the cpm genes or produce small capsids. SaPIbov2 is 27 kb tion to their carriage of important genes—interference with helper in length, owing to the presence of a 12-kb transposon, and could phage reproduction, which could ensure their transferability and not be accommodated by small capsids, whose size is presumably long-term persistence. determined by capsomere geometry. Nevertheless, SaPIbov2 strongly interferes with helper phage reproduction, blocking pla- temperate phage | small terminase | capsid morphogenesis que formation and causing a 500-fold reduction in helper phage plaque-forming titer (Table 1 and Fig. 1A; note that interference is assessed as a reduction in phage titer or as a reduction in taphylococcal pathogenicity islands (SaPIs) are prototypes plaque size or number, and that these two parameters do not of a large family of phage-inducible chromosomal islands S always match precisely). An earlier study hinted that gene 12 of (PICIs; formerly referred to as PRCIs) in Gram-positive bacteria the closely related 15-kb SaPIbov1 might be involved in phage (1), and their are organized to take optimal advantage interference (9). We originally designated this gene pif (phage of the temperate phages that they parasitize to carry out their life fi fi interference function) (9), but because pif had been used pre- cycle (1). Speci cally, SaPIs reside at speci c sites in the chro- viously (14), we now use ppi for phage packaging interference mosome of their host and remain quiescent under the control of (see below) and add a subscript to indicate its origin, e.g., ppispb2 a master repressor (2). Upon superinfection by certain phages or denotes the SaPIbov2 version. We cloned ppi gene and tested fi spb2 upon SOS induction of a resident , SaPIs use speci c its role in phage interference. As shown in Fig. 1A, the cloned nonessential phage proteins to lift the repression, enabling the gene completely blocked 80α plaque formation, and its deletion expression of their integrase and excision genes (3–5); following fully restored plaque formation and increased the 80α plaque excision, they initiate replication at a SaPI-specific replication titer nearly to that seen with the host strain lacking any SaPI origin using SaPI-coded initiator and primase, and continue replication using the host cell replication machinery (6, 7). SaPIs

reorganize the phage virion proteins to form small capsids Author contributions: G.R., C.U., J.R.P., G.E.C., and R.P.N. designed research; G.R. and K.K. commensurate with their genomes (usually about one-third the performed research; J.C., P.K.D., and K.D.L. contributed new reagents/analytic tools; G.R., size of the phage ), and they use a SaPI-coded terminase J.C., K.K., H.F.R., C.U., P.K.D., K.D.L., and R.P.N. analyzed data; and H.F.R. and R.P.N. wrote small subunit to package their DNA preferentially (8, 9). This the paper. process results in the efficient production of SaPI-specific in- The authors declare no conflict of interest. fectious particles by means of which they are spread not only *This Direct Submission article had a prearranged editor. among staphylococci but also to other bacteria, such as Listeria 1Present address: Department of Genomics and Health, Center for Advanced Research in monocytogenes (10), at very high frequency. Public Health, Valencia 46020, Spain. Upon discovery of the first SaPI, SaPI1, it was immediately 2To whom correspondence should be addressed. E-mail: [email protected]. α realized that reproduction of its inducing phage, 80 , was greatly This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. diminished. Indeed, plaque formation was blocked and the phage 1073/pnas.1204615109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1204615109 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 Table 1. Effect of wild-type SaPIs, SaPI mutations, and the mutations were at the same site, Q132, and in each of these, overexpressed SaPI genes on phage 80α and SaPI titers the glutamine was replaced by either an arginine or a lysine (Fig. S1B). The 10th mutant had an isoleucine substitution for thre- Phage titer onine 106 in the same gene. To confirm that the TerS mutations SaPI (pfu/mL) × SaPI titer, P † ‡ were necessary and sufficient to confer the observed phenotype, SaPI mutation Plasmid 108 TU/mL we replaced the native TerSP gene with the mutated one (Q132K) α 520 ± 10 in an 80 prophage and found that the mutant phage was Ppispb2 pCN51 440 ± 60 resistant (Fig. S2A). We next performed a bacterial two-hybrid α pCN51-ppispb2 2 ± 0.2 test, comparing the WT 80 TerSP with a Ppispb2-resistant TerSP cpmAB ± for any interaction with Ppispb2. As shown in Fig. 2A, Ppispb2 pCN51- sp1 15 2 α SaPIbov2 – 1 ± 0.1 1.1 ± 0.1 × 106 bound strongly to the WT 80 TerSP but not to the mutant Δppi 360 ± 10 0.5 ± 0.1 × 106 protein, and it did not interact with the cognate SaPI TerSS. Interaction between Ppi and TerS was confirmed by a pull- Δppi pCN51 230 ± 30 4.2 ± 0.4 × 106 spb2 P Δppi ppi ± ± × 6 down assay using S-tagged Ppispb2 (Fig. 2 B and C), suggesting pCN51- spb2 1 0.5 3.6 0.4 10 that Ppi directly and specifically interacts with TerS and SaPI1 – 40 ± 6 2.3 ± 0.4 × 108 spb2 P Δ ± ± × 8 hence blocks the packaging of phage DNA. Because the muta- cpmAB 250 30 3.4 0.6 10 tions were in the C-terminal region of the protein, which, in other Δ ± ± × 8 cpmAB pCN51 210 20 1.0 0.2 10 phages, is the region of binding to TerL (15, 16), we suggest that ΔcpmAB cpmAB ± ± × 7 pCN51- sp1 8 1.4 6.3 0.3 10 Ppi acts by interfering with the interaction of TerS with TerL, 8 spb2 P SaPIbov1 – 49 ± 7 4.2 ± 0.1 × 10 and thus blocks cleavage and packaging of phage DNA. Δppi 150 ± 20 1.1 ± 0.1 × 108 The TerSP proteins fall into seven superfamilies (18). Four ΔcpmAB ± ± × 9 36 7 1.6 0.3 10 tested phages were sensitive to Ppispb2 (Table S1); all of these Δ ± ± × 9 ppi, 210 80 1.2 0.2 10 belong to the terminase_2 superfamily, as do the SaPI TerSS ΔcpmAB (Fig. S2B). We tested four other staphylococcal phages that – ± ± × 8 SaPI2 3 0.4 2.9 0.3 10 encode TerSP proteins not belonging to the terminase_2 super- 8 Δppi 3 ± 0.1 5.8 ± 0.6 × 10 family, and all four were resistant to Ppispb2 (Table S1), and were ΔcpmAB{ 13 ± 1 2.0 ± 0.3 × 109 quite dissimilar in the region surrounding position 132 (Fig. S2B). 8 Δppi, 110 ± 10 3.1 ± 0.5 × 10 The terminase_2 superfamily phage TerSP proteins lack a con- { ΔcpmAB served C-terminal extension of ∼26 amino acids found in the SaPI SaPIbov5 – 390 ± 20 4.7 ± 0.2 × 106 TerSS proteins. A test of the possibility that this extra segment is responsible for the indifference of SaPI TerSS to Ppispb2 is Values are means ± SD of three independent samples (n = 3). Entries in in progress. bold type represent situations showing interference. We considered three- All known SaPIs have a Ppi homolog that belongs to one of fold or greater reduction in phage titer as interference. two conserved subsets, either SaPIbov2-like (high similarity to †Phage titer of lysate using RN4220 as indicator. pfu, plaque forming units. ‡ Ppispb2) or SaPI1-like (30% or less similarity to Ppispb2), as shown SaPI titer of lysate using RN4220 as recipient. TU, SaPI Transducing Units. { in Fig. S3A. Within the SaPIbov2 subset, ppispb2 is the strongest ORF13 deleted along with cpmAB because just cpmAB deletion could not inhibitor of 80α, ppi is weaker, and ppi is weakest (Fig. S3B), be generated. spb1 sp2 suggesting the involvement of one or more additional SaPI genes in SaPIbov1 and SaPI2 interference. The ppi genes in the SaPI1 α (Table 1). This phenotype was fully complemented by the cloned subset had no inhibitory effect on phage 80 or 80 (Fig. 1A and Fig. S4A), but the cloned ppisp1 blocked plaque formation by ϕ12 gene (Fig. 1A). Because the deletion of ppispb2 had no significant effect on the SaPIbov2 life cycle (Table 1), we suggest that Ppi, (Fig. S4B), presumably by inhibiting TerSP of the terminase_4 a SaPI-coded protein, is dedicated to phage interference. superfamily, which includes the Panton-Valentine leukocidin- We next attempted to identify the stage in the phage re- encoding Staphylococcal Leukocytolytic Toxin phages. Because SaPI1-mediated interference with 80α does not involve production cycle that was inhibited by Ppi . A test by quanti- spb2 ppi , we considered the possibility that capsomere diversion was tative real time-PCR (qPCR) for inhibition of phage replication sp1 entirely responsible for interference. Accordingly, we deleted the showed that phage replication was unaffected (Fig. S1A), sug- two principle capsid morphogenesis genes, cpmA and cpmB,of gesting that Ppispb2 was targeting a later stage. Although Ppispb2 SaPI1 and tested this deletion for its effects on 80α plaque for- blocked plaque formation, it did not prevent phage-induced lysis, mation and phage titer. As shown in Fig. 3A and Table 1, this in- indicating that the block was between replication and lysis. frame deletion virtually eliminated interference with 80α. Ad- Accordingly, we compared the particles present in a standard ditionally, when cloned and overexpressed, the two genes to- phage lysate with particles present in the lysate of Ppispb2-inhibited α α gether totally eliminated plaque formation and greatly reduced phage 80 . A lysate generated by growth of phage 80 on a con- the phage titer (Fig. 3A and Table 1). To demonstrate the basis trol strain contained normal intact phage particles (Fig. 1B), and of this effect, we analyzed the DNA present in both large- and one generated on a strain expressing ppispb2 contained mostly small-headed particles. As shown in Fig. 3C, in the absence of phage tails and proheads (Fig. 1C), indicating that phage matu- any SaPI, the particle DNA migrates as a 45-kb band, corre- ration was affected. Because proheads do not acquire tails unless sponding to monomeric phage DNA from large-headed particles. fi they have been lled with DNA (12), it seemed likely that Ppispb2 However, in the presence of SaPI1 or SaPI2, most of the particle inhibits the packaging of helper phage DNA. DNA migrates as an 18-kb band, corresponding to monomeric Packaging is initiated by the two-subunit terminase, which rec- SaPI DNA from small-headed particles, and only a tiny fraction ognizes and specifically cleaves target DNA (15–17). Phage and migrates as 45-kb DNA from large-headed particles, accounting SaPI both use the phage-encoded large (TerL) subunit, and this for the observed reduction in phage titer. We have previously is complexed with a phage- or SaPI-encoded small subunit (TerSP shown by Southern blotting of such a gel that both of these bands fi or TerSS, respectively), which determines packaging speci city contain both phage and SaPI DNA, and that most of the phage (9, 12). It therefore seemed likely that TerSP would be the target DNA migrates with the SaPI monomers (9). As also shown in of Ppispb2 inhibition. To test this, we isolated phage mutants able Fig. 3C, deletion of cpmAB eliminates the SaPI monomer-sized to form plaques on a strain containing the cloned ppi . Mutants material, all of the particle DNA migrating as a 45-kb band, − spb2 were readily obtained, at a frequency of ∼10 6, and we sequenced which includes both phage- and SaPI-specific DNAs. Therefore, the phage small-terminase subunit of 10 of these. In each case although packaging is sequence-specific and determined by TerS, there was an amino acid substitution in TerSP; in nine of these, it is not size-specific: both phage and SaPI DNAs are packaged

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1204615109 Ram et al. Downloaded by guest on September 24, 2021 MICROBIOLOGY

Fig. 1. Ppi interference with phage 80α.(A) Approximately 108 bacteria were infected with phage 80α, plated on phage bottom agar, and incubated 36–48 h

at 32 °C. Plates were stained with 0.1% TTC in TSB and photographed. Genes cloned into pCN51 were induced with 1.0 μM CdCl2.(B and C) Electron mi- croscopic analysis of sedimented particles from (B) phage 80α lysate of RN4220 + pCN51 and from a (C)80α lysate of RN4220 + pCN51 – ppispb2. CP, complete phage 80α particles; FT, free phage tails; PH, phage proheads.

in capsids of both sizes. Because the complete SaPI genome is determined capsid morphogenesis results in the packaging of packaged in capsids of either size, capsid remodeling has relatively subgenomic and therefore defective phage DNA segments. These little effect on most SaPI titers. Because the complete phage results suggest that there are at least two different mechanisms genome cannot be accommodated in the small capsid, CpmAB- of SaPI-mediated interference with helper phages: Ppi-mediated

Fig. 2. Interactions between Ppispb2 and TerSP (80α). (A) BACTH analysis. Spots in each row represent three independent colonies. Plasmid combinations are numbered as follows: 1, pKT25-zip + pUT18C-zip

(positive control); 2, pKT25-TerSP (WT80α) + pUT18- Ppispb2; 3, pKT25-TerSP (Ppispb2-resistant 80α) + pUT18-Ppispb2; 4, pKT25-TerSS (SaPIbov2) + pUT18- Ppispb2; and 5, pKT25 + pUT18 (negative control). Blue color indicates cAMP-dependent lacZ expres- sion following reconstitution of adenlyate cyclase activity by interaction of fusion proteins. (B and C)

Pull-down of His-tag–TerSP (80α) coexpressed with Ppispb2–S-tag using S-protein agarose column: lane 1, protein eluted from S-protein agarose beads after binding and washing of cell lysate obtained from cells coexpressing both the proteins; lane 2, pre- stained markers; lane 3, eluate from S-protein aga- rose beads after binding and washing of cell lysate

obtained from cells expressing His-TerSP (80α); lane 4, cell lysate obtained from cells expressing His-TerSP (80α). (B) Coomassie-stained gel. (C) Western blot of the gel shown in B using Penta-His HRP conju- gate and S-protein HRP conjugate antibodies.

Ram et al. PNAS Early Edition | 3of6 Downloaded by guest on September 24, 2021 Fig. 3. Effect of SaPI interference genes on phage 80α. Phage platings were as in Fig. 1A.(A) Effect of SaPI1 CpmAB. (B) Effects of SaPI2 and SaPIbov1 CpmAB and Ppi. Note that ORF13 was always included in deletions of SaPI2 cpmAB because a deletion of the two genes alone could not be constructed. ORF13 does not affect interference. (C) Agarose gel analysis of DNA extracted from sedimented phage particles. Phage 80α was grown on RN4220 and various derivatives of SaPI2 (i; Upper) or SaPI1 (ii; Lower). The resulting lysates were treated with DNase, PEG-precipitated, and phenol extracted. Equivalent amounts of DNA were separated on 0.8% agarose gel for 13 h at 60 V, strained with ethidium bromide, and photographed.

interference with packaging and Cpm-mediated diversion of with it, having no effect on either 80α plaque formation or phage capsomere proteins for small capsid formation. titer (Fig. S4C and Table 1). However, the yield of SaPIbov5 Because ppi could account for at most a part of the observed transducing units (SaPI TU) is four orders of magnitude lower interference with 80α by SaPIbov1 or SaPI2 (Figs. 1A and 3B than the phage titer, in contrast to SaPI titers within 1–2 orders and Table 1), we considered the possibility that cpmAB and/or of magnitude of the helper phage seen for SaPIs that interfere other SaPI genes might also be involved. With SaPIbov1 and with 80α. If interference is important for SaPI survival, we pre- SaPI2, this was, indeed, the case, because both cpmAB and ppi dicted that SaPIbov5 would survive by interfering with other ϕ are involved. On one hand, cpmABsp2 completely blocks plaque helper phages, and, indeed, it interferes with 11 (Table 2 and formation in the absence of ppisp2, and ppisp2 blocks plaque Fig. S4D), but not via ppispb5 (Fig. S5A), perhaps using the same formation in the absence of cpmABsp2, suggesting that the two interference mechanism as that used by SaPI2 against phage 80. interferences are redundant. On the other hand, ppispb1 has This interference seems paradoxical, however, because SaPIbov5 ϕ a much stronger effect than cpmABspb1, and the two mechanisms virtually eliminates plaque formation by 11 but causes only appear to be additive; however, they do not fully account for the a ∼3× decrease in phage titer. This apparent paradox was re- observed interference with 80α by SaPIbov1, suggesting that there solved by a one-step phage growth analysis, as shown in Fig. S5B. may be yet a third mechanism. Although SaPIbov1 deletions and Although the reduction in burst size paralleled the reduction in subclones have not been very informative in this respect, similar overall phage titer, lysis was delayed by ∼15 min. Because phages studies with SaPI2 clearly demonstrate the existence of a third multiply and form plaques only during growth of the indicator interference mechanism. For these experiments, we used phage bacteria, plaque size is profoundly affected by burst size and la- 80 rather than 80α, because phage 80 does not support the tency, and we suggest that this combination of effects is sufficient production of small SaPI particles (19) and is not inhibited by to account for the observed effect of SaPIbov5 on ϕ11 plaque fi any of the ppi genes described above or by cpmABsp2 (Fig. S4A formation. Although not tested speci cally for this report, we and Fig. 4). Nevertheless, SaPI2 inhibits phage 80 very strongly, suggest that other examples of disparity between phage titer and completely blocking plaque formation (Fig. 4). This inhibition plaque size can probably be similarly explained. was largely eliminated by deletion of SaPI2 ORF17, which has homologs in the four other SaPIs analyzed in this study. When Discussion expressed from the exogenous promoter Pcad, ORF17 blocks Interference with phage propagation has been observed in a va- plaque formation by phage 80 but has no effect on 80α, whereas the riety of situations and by a variety of mechanisms (aside from cloned SaPI2 ppi and cpmAB genes interfere with phage 80α but classical superinfection immunity and restriction modification). have no effect on phage 80 (Fig. 4). These results clearly dem- Among these strategies are interference with late phage T7 onstrate three independent mechanisms of phage interference and mRNA translation by the pifA and pifB genes of plasmid F (14), show additionally that the interference is largely phage-specific. mutual interference between λ and P2, owing to inactivation of According to these results, one would predict the existence RecBC by λ (20), and interference with several phages by SP02, of SaPIs that use only that third mechanism. A possible example possibly by a similar mechanism (21); a rather artificial mecha- of this is a recently identified SaPI, SaPIbov5, which lacks cpm nism is interference with one phage by the cloned homolog of genes and contains a ppi gene of the SaPI1 type, which does not one of its essential genes (22) or by phage genome fragments affect 80α. SaPIbov5 is induced, packaged, and transduced at (defective interfering particles) (23). A hitherto unknown in- high frequency by phage 80α, but does not significantly interfere terference mechanism is the incorporation during phage infection

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1204615109 Ram et al. Downloaded by guest on September 24, 2021 a universal feature of the PICIs, such as the SaPIs. Moreover, a tBLASTn search with prototypes of either of the Ppi classes identifies only the Ppi homologs in the PICIs plus hypothetical proteins in pathogenicity islands (probably PICIs) of a few other Gram-positive organisms—Eubacterium limosum KIST612, Aer- ococcus viridans ATCC 11563, Clostridium bolteae ATCC BAA- 613. Thus, the Ppi mechanism of phage interference may be unique to such elements. A key SaPI feature that sharply differentiates it from its helper phages is the presence of a SaPI-coded TerSS, which differs from the phage TerSP by a 26-residue C-terminal extension. Ppispb2 binds to the phage small-terminase subunit TerSP,specifically blocking the packaging of phage DNA (Figs. 1C and 2); it does not bind the SaPI TerSS (Fig. 2A). The precise mechanism of this interference has yet to be determined; one possibility is that Ppispb2 binding to TerSP could abolish its DNA binding activity; alternatively, it could prevent the binding of TerSP to TerL. Tests to distinguish between these possibilities and to determine whether the C-terminal extension of TerSS is responsible for Ppispb2 resistance are in progress. We have also found that SaPIs use at least two other mech- anisms of phage interference: one resulting from the diversion of capsid proteins to the formation of small, SaPI-sized capsids, and the other resulting from the expression of SaPI2 ORF17, whose mechanism remains to be determined. Each of the five different SaPIs studied here interferes with at least one of the four tested. Interference with infecting bacteriophages is a widespread

strategy that has evolved primarily or exclusively to benefit host MICROBIOLOGY bacteria (14, 20–25). Here, however, we have a tripartite system consisting of host, SaPI, and phage, of which the first two clearly share in the benefits, and we suspect that the third does also because interference is so widespread and well-conserved. The importance of interference is underlined by the fact that SaPIs have evolved multiple mechanisms (Table S2). The question then arises of why SaPIs interfere with helper phage reproduction. One possibility is that interference helps the SaPI to keep pace with the phage during successive infection cycles. As noted, there is a vast excess of SaPI monomer-sized DNA in particles isolated from a typical WT SaPI1 or SaPI2 lysate, but not from a cpmAB mutant (Fig. 3C). Thus, SaPI1 and SaPI2 cause the phage to waste much of its DNA by dead-end packaging in small capsids—averyneat way for the SaPI to ensure that it would keep pace with the phage. We note additionally that, as is typical of bacteriophages, virion proteins (and therefore capsids) are in great excess so that SaPI and phage do not automatically compete for packaging. In other words, without a specific interference mechanism, SaPI matura- tion does not adversely affect phage maturation, as can be seen in Fig. 4. SaPI2 interference with different phages. SaPI2 uses three mecha- Table 1. A second major advantage could be a reduction in the nisms of phage interference: with phage 80 (Left) it uses ORF17, and with probability that an organism receiving a SaPI will be killed by phage 80α (Right) Ppi and CpmAB. a coinfecting phage; this would enhance the survival of SaPIs in the population (although it would not be reflected in the single- round assays reported here). A third major advan- of short segments of phage DNA by chromosomally located tage is that an interfering SaPI sharply increases the frequency of (clustered regularly interspaced short palindromic host cell survivors that are not lysogens (11). repeats) that are used to prime synthesis of interfering small Fascinating questions on the evolutionary history of the SaPIs RNAs that block phage development (24, 25). In this study we are raised by these studies. Why don’t helper phages avoid the describe a unique mechanism of phage interference in which phage DNA packaging is blocked by Ppi. All SaPIs sequenced to date encode a Ppi homolog (1) and presumably use this mecha- Table 2. Effect of SaPIbov5 on phage ϕ11 and SaPI titers ’ nism of phage interference. Two classes of Ppi shavebeeniden- SaPI Phage titer SaPI titer, fi † ‡ ti ed by sequence analysis and have been shown by genetics to SaPI mutation Plasmid (pfu/mL) × 108 TU/mL interfere correspondingly with two different sets of phages. Class I (prototype SaPIbov2) interferes with phages 80α and ϕ11, and 140 ± 10 class II (prototype SaPI1) interferes with ϕ12. No SaPI encodes SaPIbov5 49 ± 5 3.8 ± 0.6 × 107 more than one Ppi; however, SaPIs may use other interference Values are means ± SD of three independent samples (n = 3). Entries in mechanisms in lieu of or in addition to the Ppi-mediated one for fi bold type represent situations showing interference. We considered three- certain phages, and these mechanisms may be speci c for dif- fold or greater reduction in phage titer as interference. † ferent phages. Each of the five SaPIs studied here interferes with Phage titer of lysate using RN4220 as indicator. pfu, plaque forming units. at least one known phage; therefore, phage interference may be ‡SaPI titer of lysate using RN4220 as recipient. TU, SaPI Transducing Units.

Ram et al. PNAS Early Edition | 5of6 Downloaded by guest on September 24, 2021 SaPI-mediated interference either by mutating the antirepressor Mutant Construction. In-frame deletion mutants were generated using the allelic exchange vector pMAD (29) as described previously (2). Details are protein (3) or by mutating TerSP? For the 27-kb, transposon- carrying SaPIbov2, which came first: acquisition of the transposon given in SI Materials and Methods. or loss of the cpm genes? Assuming that the C-terminal extension Phage Infection and Induction. strains (in triplicates) were in the TerSS proteins is responsible for Ppi resistance, was it ac- inoculated in CYGP (Casamino acids Yeast extract Glycerophosphate) broth at an quired along with or before the acquisition of ppi, and what was its 8 initial OD600 = 0.1 (∼1 × 10 cfu/mL) and grown at 37 °C and 225 rpm. Cultures source, or that of ppi? Is the Ppi mechanism of interference used 9 were adjusted to OD600 = 1.0 (∼1 × 10 cfu/mL) with CYGP broth and diluted in other contexts in the bacterial world? Implicit in these questions α is the view that SaPIs are independently evolving entities that 1:1 with phage buffer, infected either with phage 80 at a multiplicity of have “borrowed” certain phage genes but explicitly excluded infection (MOI) of 3 or with phage 80 at an MOI of 2. Induction methods are others. This behavior provides them with an optimal existence as detailed in SI Materials and Methods. satellites/parasites, which they have fine-tuned by developing multiple interference functions that target different helper phages. Plaque Formation Assay on Different SaPI-Containing Strains. Known number of phage particles e.g., phage 80α (∼200–300 phages), phage 80 (∼300 phages), phage Φ11 (∼500 phages), and Φ12 (104 phages) were mixed with ∼108 cells Materials and Methods 9 (100 μL culture adjusted to OD600 = 1.0 ∼1 × 10 cfu/mL) of RN4220 and Strains and Plasmids. Bacterial strains, plasmids, and primers used in this study its derivative strains. This mixture was incubated at RT for 15 min, sub- – are listed in Tables S3 S5. Growth media (26) and conditions are described sequently mixed with 3 mL of phage top agar (26) and immediately Φ α in SI Materials and Methods. Staphylococcal temperate phages 80, 80 , poured on phage bottom agar (26) plate with or without 1.0 μM CdCl . Φ Φ Φ Φ Φ Φ 2 11, 12, 55, 187, NM1, and NM4 were used in this study. Plates were incubated at 32 °C for 36–48 h and stained with 0.1% TTC (Difco) in TSB broth (30). Molecular Methods. All general DNA manipulations (i.e., digestion, ligation, etc.) were carried out by standard methods (27). Oligonucleotides used for Phage and SaPI Titer Measurement. Phage lysates with or without SaPI were this work are listed in Table S5. All restriction enzymes and T4 DNA ligase filter sterilized and serially diluted in phage buffer (26). Please see SI Materials were purchased from New England Biolabs. Primers were obtained from and Methods for further details. Integrated DNA Technologies Inc., and DNA sequencing was performed by Macrogen. Electron Microscopy, Bacterial Two-Hybrid Assays, and qPCR. Please see SI Materials and Methods. Cloning, Expression, and Pull-Down Assay. Escherichia coli BL21 (DE3) cells were transformed with plasmid pGR00020 and plasmid pGR00021 for ex- ACKNOWLEDGMENTS. We thank Eric W. Roth for his support and help with pression analysis. Plasmid pGR00020 expresses the TerSP protein fused with electron microscopy at New York University Langone Medical Center, Office – α His-tag [His-tag TerSP (80 )], and plasmid pGR00021 coexpresses the His- of Collaborative Science Microscopy Core facility. This work was supported α – TerSP (80 ) as well as the Ppispb2 protein fused with S-tag (28) (Ppispb2 S-tag). by National Institutes of Health Grants R01AI022159 (to R.P.N. and J.R.P.), See SI Materials and Methods for further details. and R21 AI067654 and R56 AI081837 (to G.E.C.).

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