Precisely modulated interference with late phage gene transcription

Geeta Ram, John Chen, Hope F. Ross, and Richard P. Novick1

Skirball Institute Program in Molecular Pathogenesis and Departments of Microbiology and Medicine, New York University Medical Center, New York, NY 10016

Edited by James M. Musser, Houston Methodist Research Institute, Houston, TX, and accepted by the Editorial Board August 16, 2014 (received for review April 16, 2014) Having gone to great evolutionary lengths to develop resistance and, perhaps to gain an advantage, SaPIs interfere with the to , bacteria have come up with resistance mech- reproduction of their helper phages (7). Thus far, two distinctly anisms directed at every aspect of the life cycle. different SaPI-coded interference mechanisms have been iden- Most genes involved in phage resistance are carried by plasmids tified, namely the capsid morphogenesis genes cpmA and B, and other , including bacteriophages and which cause the formation of small capsids commensurate with their relatives. A very special case of phage resistance is exhibited the SaPI , and the phage packaging inhibition gene ppi, by the highly mobile phage satellites, staphylococcal pathogenic- which blocks phage terminase small subunit (TerSP), favoring the ity islands (SaPIs), which carry and disseminate superantigen and packaging of SaPI DNA. ppi is present in all of the ∼50 SaPI other virulence genes. Unlike the usual phage-resistance mecha- analyzed thus far; cpmAB is present in most, but not nisms, the SaPI-encoded interference mechanisms are carefully all. Both are encoded by and are functional in three pro- crafted to ensure that a phage-infected, SaPI-containing cell will totypical SaPIs, SaPI1, SaPI2, and SaPIbov1. lyse, releasing the requisite crop of SaPI particles as well as a In studies of SaPI interference with helper phage 80 (of the greatly diminished crop of phage particles. Previously described international typing set, a member of the famous 80/81 pair) (8), SaPI interference genes target phage functions that are not which is related to but is distinctly different from the more ex- required for SaPI particle production and release. Here we describe tensively studied 80α, we observed that SaPI2, the major cause of a SaPI-mediated interference system that affects expression of late menstrual toxic shock, inhibits phage 80 reproduction more phage gene transcription and consequently is required for SaPI MICROBIOLOGY and phage. Although when cloned separately, a single SaPI gene stringently than do other SaPIs, and preliminary results sug- totally blocks phage production, its activity in situ is modulated gested that SaPI2 uses a third interference mechanism (7). In accurately by a second gene, achieving the required level of this report we characterize this third interference mechanism interference. The advantage for the host bacteria is that the SaPIs used by SaPI2 against phage 80 and other related phages but not α curb excessive phage growth while enhancing their gene transfer against phage 80 . activity. This activity is in contrast to that of the clustered regularly Results interspaced short palindromic repeats (), which totally block phage growth at the cost of phage-mediated gene transfer. Identification of the SaPI2-Coded Inhibitor(s) of Phage 80. To de- In staphylococci the SaPI strategy seems to have prevailed during termine whether SaPI2 uses the same interference mechanisms evolution: The great majority of strains against phage 80 as it does against phage 80α, we cloned the carry one or more SaPIs, whereas CRISPRs are extremely rare. cpmAB and ppi loci of SaPI2 separately to the vector pCN51 (9), behind the exogenous cadmium-inducible promoter (Pcad) (10) helper phage | transcription regulation | bacteriophage resistance and tested these clones for inhibition of phage 80. Although either of these clones completely blocked plaque formation by he staphylococcal pathogenicity islands (SaPIs) are pro- Ttotypical members of a increasingly recognized family of Significance highly mobile ∼15-kb phage-inducible chromosomal islands, which were discovered serendipitously because of their carriage of the Highly mobile staphylococcal pathogenicity islands (SaPIs) are gene for toxic shock syndrome toxin-1, tst, of which the SaPIs are the only source of toxic shock toxin and certain other super- the only source (1). The SaPIs are intimately related to certain antigens, especially enterotoxin B. To promote their survival helper phages whose life cycles they parasitize to enable their and spread, the SaPIs parasitize and interfere with certain propagation and spread. bacteriophages. Unlike the interference of the clustered regu- SaPIs exist quiescently at specific chromosomal sites under the larly interspaced short palindromic repeats (CRISPRs), the in- control of a master repressor. A defining connection between terference of SaPIs is never complete, allowing horizontal gene the helper phage and the SaPI lifestyle is induction by helper transfer and adaptation. We report a novel SaPI-determined phage-encoded antirepressor proteins (2). These proteins lift interference mechanism that targets a phage gene essential for the repression, setting in motion the SaPI life cycle: excision, both phage and SaPI. Because SaPI is not self-destructive, it replication, and encapsidation of SaPI DNA into infectious must modulate this inhibition to ensure production of its own phage-like particles. The resulting SaPI transfer frequencies infectious particles, as well as those of the phage, and it does approach the pfu titer of the helper phage (3). A key SaPI fea- so by means of a novel SaPI protein that binds to the inhibitor. ture is that the SaPIs encode homologs of the phage terminase “ ” Author contributions: G.R. designed research; G.R. performed research; G.R., J.C., and small subunit, referred to herein as TerSS, that recognize a R.P.N. contributed new reagents/analytic tools; G.R., J.C., H.F.R., and R.P.N. analyzed specific SaPI packaging (pac) site to initiate packaging into data; and H.F.R. and R.P.N. wrote the paper. proheads composed of helper phage virion proteins (4, 5). The The authors declare no conflict of interest. phage and the SaPI TerS do not cross-react so that packaging is This article is a PNAS Direct Submission. J.M.M. is a guest editor invited by the Editorial DNA-specific (6). Board. The SaPI life cycle is initiated only when the life cycle of a 1To whom correspondence should be addressed. Email: [email protected]. helper phage is in progress; SaPI particle production, however, is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. not quite able to keep up with helper phage maturation rate, 1073/pnas.1406749111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1406749111 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 phage 80α (7), we were surprised to find that neither, even when induced, had any effect on plaque formation by phage 80 (Fig. 1A). To confirm this effect in situ, we deleted both ppi and cpmAB from SaPI2 and found that the interference was un- affected. This result showed that there must be a third inter- ference mechanism; to identify it, we cloned each of the SaPI2 ORFs separately, using the same vector, and found that one of the ORFs, 17, caused interference. ORF17, when cloned, blocked plaque formation by phage 80 (Fig. 1A) (7). One other ORF, ORF13, was toxic to the host cells and could not be expressed adequately from a plasmid. It was found to cause in- terference on the basis of a deletion (see below). ORF13 and -17 are located in operon 1, which includes cpmA and B, ORF16, and terSS. ppi is upstream of the operon and is equivalent to ORF10 (Fig. 1B). No other SaPI2 gene had any detectable inhibitory effect on phage 80.

ORF17-Mediated Interference. We began with ORF17 because it could be overexpressed. To identify the phage gene(s) targeted by ORF17, we isolated ORF17-resistant mutants as rare plaques Fig. 2. (A) Comparison of the rinA and ltrC genomic region from 80α and on a strain containing the cloned and overexpressed ORF17. 80. Note that the position of ltrC (ORF32) in phage 80 corresponds to that of Thirteen of the mutant phages were sequenced. All 13 had point rinA in phage 80α. “P” represents the late gene promoter. (B) Activation of mutations in a single gene, 32, and all had amino acid replace- phage 52A late transcript promoter by LtrC. The late transcript promoter β ments at a single site in gp32: tyrosine 67 was replaced by his- was cloned to plasmid pCN41 as a transcriptional fusion to -lactamase. The resulting reporter plasmid was introduced into strains lysogenic for either tidine in 11 mutants and by cysteine in two mutants (Fig. S1). the WT or a derivative prophage containing a deletion of ltrC in The sequence of gene 32 showed it to be late transcriptional the absence or presence of SaPI2. β-Lactamase activity was measured 2 h regulator C, ltrC (11, 12), which corresponds to 80α rinA (13). after induction with mitomycin C (MC) and in a parallel culture without in- LtrC and RinA are regulatory proteins that activate the tran- duction. (C) Effects of cloned ptiA and ptiM on the phage late transcript scription of the late phage operon (morphogenetic and lysis promoter. ptiA and ptiM were cloned singly or together to pCN51, and their genes) (Fig. 2A). Five families of these proteins have been de- effects on LtrC activation of the late transcript promoter–β-lactamase fusion scribed, constituting a superfamily of late transcriptional regu- were determined. For these tests, the pti genes were induced with 1.0 μm β lators (Ltr) (11, 12). These are essential phage genes (14, 15) CdCl2,the -lactamase fusion construct was integrated in the chromosome corresponding to coliphage lambda gene Q. To test phage 80 at the SaPI4 att site (lab plasmids collection), and LtrC was provided by su- perinfection with phage 80. Cultures were assayed at 2 h postinfection, and LtrC for its role in transcriptional activation, we needed to delete assays were performed in triplicate. the gene. Because the gene is presumed to be essential for phage reproduction, this deletion would have to be done with the prophage. This deletion could not be done with phage 80, which phage 80. Using a phage 52A lysogen, we introduced an in-frame cannot form a lysogen (13), so we turned to a close relative, deletion in ltrC and found that the phage, as predicted, was phage 52A, which can form lysogens, can induce SaPI2, and unable to lyse the cell or to produce any infectious phage par- whose LtrC and late gene operon are identical to those of ticles. We next cloned the β-lactamase reporter downstream of the late phage promoter in plasmid pCN41 in RN450 with either the WT or ltrC mutant 52A prophage. The ltrC mutant did not detectably activate the late gene promoter (Fig. 2B); following mitomycin C treatment, the culture did not undergo lysis, and no detectable phage were produced, confirming the essentiality of the gene for phage 52A and, by inference, for phage 80. The presence of SaPI2 had no effect on the β-lactamase activity in the strain with the ltrC mutant but, as predicted, had a profound effect on the activity in the strain with the WT phage (Fig. 2B). Using the WT phage, we next demonstrated inhibition of late phage transcription by the cloned SaPI2 ORF17. These results are presented in Fig. 2C and have suggested the designation “phage transcription inhibitor A” (ptiA) for the gene (ORF17). Because PtiA blocked expression of the entire late phage gene module, we predicted it would block lysis as well as plaque for- mation. Note that the lysis of a culture and the formation of plaques on an indicator strain are rather different tests of phage Fig. 1. (A) Phage interference mediated by cloned SaPI2 genes. The in- function: Plaque formation can be blocked by a modest re- dicated genes were cloned to plasmid pCN51 under a cadmium-inducible promoter (Pcad). Strain RN4220 containing the indicated plasmids was duction in burst size, whereas lysis can be blocked only by pre- infected with phages 80α (∼150 pfu per plate) or 80 (∼250 pfu per plate) venting the expression of the lysis module. Unlike the other two μ plated on phage bottom agar containing 1.0 MCdCl2 and incubated for 48 h interference mechanisms, which blocked plaque formation but at 32 °C. Plates were stained with 0.1% TTC in TSB and photographed. (B) not lysis (7), PtiA, as predicted, blocked both (Fig. 3A), making it Map of SaPI2 operon 1. Genes previously shown to cause phage interference are shown in red; hypothetical genes of unknown function are shown in the most severe of the interference mechanisms yet identified. blue; numbered ORFs newly determined to be involved in interference are Also, predictably, the cloned ptiA blocked plaque formation by shown in orange; and the SaPI2 small terminase subunit is shown in green. other phages encoding LtrC, as shown in Fig. S2.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1406749111 Ram et al. Downloaded by guest on September 24, 2021 Modulation of PtiA Activity. As noted above, when overexpressed (cloned to a plasmid and driven by an inducible promoter), PtiAS2 not only blocks phage 80 plaque formation but totally prevents lysis (Fig. 3A). The logic of SaPI-phage parasitism would indicate that lysis prevention cannot be the normal effect of PtiAS2 because it would be suicidal for the SaPI as well as for the phage. However, SaPI2 is not a suicide bomber: Although WT SaPI2 eliminates plaque formation by phage 80, the infected cultures undergo lysis. So we next attempted to find the basis for this difference. Because, when overproduced, PtiAS2 totally blocks phage 80 reproduction, and because overproduction is expected during SaPI replication, it seemed unlikely that the difference is based simply on relative gene dosage. In fact, even in single copy, ptiAS2 has a profound effect on phage 80 re- production (Fig. S4). Therefore, we considered the possibility that PtiAS2 action is modulated by a second SaPI2 gene. As seen in Fig. 1B, ORF16 is immediately upstream of ptiA, and se- quence analysis suggested that the two genes probably are translationally coupled. Therefore, ORF16 was a likely candi- date for the putative modulator of PtiAS2. To test this possibility, we compared the effects of the cloned ptiAS2 with those of a clone containing both ORFs on the activity of phage 80. As seen in Fig. 3A, the cloned ptiAS2 totally inhibited phage 80, whereas the ORF16-ptiAS2 clone caused only modest inhibition of the phage and did not prevent lysis. Furthermore, ORF16 also re- duced the PtiAS2-mediated inhibition of the phage late gene transcription (Fig. 2C). Because the clone of ORF16 alone had little, if any, effect on plaque formation, it is suggested that MICROBIOLOGY Fig. 3. (A) Modulation of PitA activity by PtiM. (Upper) Plaque assay. Phage ORF16 is, in fact, the modulator of PtiAS2. If so, ORF16 could platings were as in Fig. 1A.(Lower) Lysis assay. RN4220strains carrying pCN51 act at the level of ptiAS2 transcription or at the level of trans- μ (vector) or carrying the pti clones used were grown with the inducer (1.0 M lation of the product, or it could form an inhibitory complex with CdCl2), infected with phage 80 at a multiplicity of infection (MOI) of 0.2, the putative protein. Because the two genes almost certainly are and incubated at 32 °C at 60 rpm for 3 h. (B) Bacterial adenylate cyclase- cotranscribed, it seemed highly unlikely that ORF16 controlled based two-hybrid (BACTH) analysis. Spots in each row represent three ptiA independent colonies. Plasmid combinations are numbered as follows: the transcription of S2. As noted above, modulation of gene

1, pKT25-zip + pUT18C-zip (positive control); 2, pKT25-LtrC (WT80) + pUT18-PtiAS2; dosage is unlikely to be the mechanism of control by ORF16. 3, pKT25-LtrC (PtiAS2-resistant 80) + pUT18-PtiAS2;4,pKT25-PtiMS2 + pUT18- Therefore, it also seemed unlikely that ORF16 controlled the + PtiAS2; 5, pKT25 pUT18 (negative control). Blue color indicates cAMP- translation of ptiAS2. Accordingly, we considered it more likely dependent lacZ expression following reconstitution of adenylate cyclase that there would be direct binding between the two proteins. We activity by the interaction of fusion proteins. tested for this direct binding by performing a second bacterial two-hybrid assay to test for possible binding of the ORF16 B Mechanism of Action of PtiA. Because all the PtiA-resistant mutations product, gp16, to PtiAS2. As shown in Fig. 3 row 4, a positive mapped in the LtrC coding sequence, it was likely that PtiA acted binding reaction was readily demonstrated, suggesting that gp16 by direct protein binding. Accordingly, we performed a two- modulates the activity of PtiAS2 by a direct binding interaction. hybrid assay, as shown in Fig. 3B. As can be seen (Fig. 3B, row 2), Because PtiAS2 has two binding partners, which could be mu- tually exclusive, the equilibrium between these partners could there was a strong positive reaction between PtiA and the LtrC account for a subtle and precise modulation system. On the basis WT protein, but no reaction was detected between PtiA and a of these results we propose that gp16 be designated “modulator PtiA-resistant mutant of LtrC (Fig. 3B, row 3). Therefore we of PtiA ,” PtiM . conclude that PtiA interferes with phage 80 growth by bind- S2 S2 To confirm the effects of PtiAS2 and PtiMS2 in the intact SaPI, ing to LtrC and directly inhibiting its ability to activate late we constructed in-frame deletions of the two genes and tested gene transcription. them individually and together for their effects on phage 80 It is interesting that the phage 80 proteins corresponding to plaque formation. As seen in Fig. 4, deletion of ptiAS2 resulted in the 80α targets of Ppi (TerS ) and of CpmAB (presumably the P small plaques; deletion of ptiMS2 eliminated plaque formation, as phage head scaffolding protein) are considerably different from expected, because of the absence of its modulatory effect on the corresponding 80α proteins and are unaffected by these ptiAS2. Also as expected, deletion of both caused the formation inhibitors. In fact, phage 80 virion proteins are not formed of small plaques indistinguishable from those seen with deletion into small capsids by any of the three prototypical SaPIs (13); of ptiAS2 alone; however, the failure of these deletions to restore α moreover, 80 is indifferent to PtiA, presumably because of the the formation of full-sized plaques is consistent with interference insensitivity of RinA to the inhibitor (Fig. 1A). PtiA, in turn, also by ORF13, because, as noted above, none of the other SaPI2 occurs in variant forms, and we have cloned those of SaPI1 and ORFs showed interference. Accordingly, we then constructed an SaPI1bov1. The SaPI1 variant PtiAS1 differs from PtiAS2 by a in-frame deletion of ORF13 and, indeed, this deletion resulted single amino acid substitution and is equally effective against in partial restoration of plaque formation (Fig. 4); deletion of phage 80; however, the SaPI1bov1 variant PtiASB1 differs at 10 both ptiAS2 and ORF13 restored plaques to full size (Fig. 4). sites and does not inhibit phage 80 (Fig. S3). PtiA homologs are More definitive results were obtained by one-step growth anal- encoded by all intact SaPIs, and their sequences suggest that they ysis of phage 80 in the presence of SaPI2 with and without fall into different groups. Perhaps PtiA variants encoded by differ- deletions of the interference genes studied here. As shown in ent SaPIs inhibit LtrC variants encoded by different phages. Fig. 5A, deletions of the various interference genes restored the

Ram et al. PNAS Early Edition | 3of6 Downloaded by guest on September 24, 2021 Fig. 4. Effects of SaPI2 ptiA, -B, and -M deletions on SaPI2 interference with phage 80. Strain RN4220 containing WT SaPI2 or various SaPI2 deletion mutants was infected with phage 80 (∼250 pfu per plate), plated on phage bottom agar, and in- cubated for 48 h at 32 °C. Plates were stained with 0.1% TTC in TSB and photographed.

growth of phage 80 to different extents, roughly paralleling the the late phage promoter, as shown in Fig. 5B (light green bar). It plaque formation tests. relieved the inhibition by SaPI2 to about the same extent as the ptiAS2 deletion (aqua bar) and, more strikingly, in conjunction ORF13-Mediated Interference. Because of its bacterial toxicity, with the ptiAS2 deletion, it fully restored promoter activity (or- Escherichia coli ORF13 could not be cloned in and could be ange bar). On the basis of these results, we have designated cloned in Staphylococcus aureus only behind a promoter (Ptet) ORF13 ptiBS2. It is likely, but not certain without mutational with an extremely low basal level of expression. Bacteria could evidence, that this is the only interference effect of PtiBS2. grow on plates only at concentrations of the inducer anhy- The question that then arises is whether PtiB is toxic to host drotetracycline (ATc) ≤2 ng/mL; growth on an ATc gradient S2 cells in its native context. In the absence of SaPI or SOS in- plate (0–20 ng/mL) is shown Fig. S5A. Plaque size was slightly duction, it would not be expressed. Because the host cells lyse larger at the borderline of inhibition, suggesting that the host cells are more sensitive to ORF13 than the phage. Thus, our after the induction of a helper phage, PtiBS2 could not be so toxic usual strategy for identifying interference targets, namely the to the host cells that it would interfere with the phage lytic cycle. tet ptiB formation of rare mutant plaques by the inhibited phage, could Indeed, after P induction in broth, the S2-containing strain B not be used in this case. Because we already had a very useful grows for six generations and then stops sharply (Fig. S5 ), β-lactamase fusion to the late phage promoter, it seemed suggesting that the synthesis of an essential protein is inhibited worthwhile to perform the extremely simple test of measuring and that there is an ∼64-fold excess of this protein in exponential the effects of the ORF13 deletion on that promoter before cells, an amount that surely is more than sufficient to support the attempting to identify the unknown phage function that was lytic cycle. Therefore, its host cell toxicity is irrelevant to phage inhibited. In fact, the ORF13 deletion had a profound effect on propagation. In principle, one could test for host inhibition in the

Fig. 5. (A) One-step phage growth analysis of ptiA- and ptiB-mediated interference of phage 80. Bacteria were infected with phage 80 at an MOI of 10, washed to remove unadsorbed phage, diluted to contain ∼104 infective centers/mL, and then incubated for 90 min. Samples were removed at the indicated times and plated for plaque formation using RN4220 as the indicator. Three replicates were used for each strain. (B) Effect of deletion of ptiA, ptiB, ptiM,and their combinations on phage 80 LtrC activity. The late phage transcript promoter–β-lactamase reporter used for the experiments in Fig. 2 B and C was tested in RN4220 with SaPI2 and several of its deletion mutants. Strains were infected with phage 80 at an MOI of 0.2. The infected cultures were incubated at 32 °C, 60 rpm and assayed for β-lactamase at 2.5 h postinfection. Three replicates were used for each strain.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1406749111 Ram et al. Downloaded by guest on September 24, 2021 absence of any phage, because SaPI operon 1, in which the gene is located, is repressed by LexA (16). Although SOS induction relieves LexA repression, the effect is transient, and transient expression of ptiBS2 would not provide a clear answer because cells would have six generations to recover from any toxicity. We suggest that, in any case, the possible toxicity of PtiBS2 is of little or no importance to the phage or SaPI life cycle. Another question that arises is whether PtiBS2, like PtiAS2,is modulated by a separate SaPI gene product. The simplest route to answering this question would be via host mutants resistant to PtiBS2. Although colonies appear on inducer-containing plates at − a frequency of ∼10 6, analysis of 25 such colonies revealed that all have mutations in Ptet. Screening of additional colonies is in progress. Alternatively, an approach involving further cloning and deletion studies has been initiated; however, that work is beyond the scope of this report. Discussion Among the remarkable abilities of bacteria to adapt to unfavorable environmental contingencies is their resistance to bacteriophages. Especially as exemplified by the lactic acid bacteria, resistance has been developed to every imaginable phase of the bacterio- phage life cycle (17). Also remarkable is that most of the bac- teriophage resistance mechanisms are encoded by plasmids and other mobile elements. Plasmids and other mobile elements seem particularly suited to the carriage and transfer of resistance genes because such resistance would not involve mutational

alterations of core functions. We suggest that phage resistance MICROBIOLOGY Fig. 6. Summary of SaPI2-mediated interference with phage 80. SaPI2 op- may well exemplify this principle, and we point to the special eron 1 encodes three proteins, PtiA, PtiM, and PtiB, involved in phage 80 case of the SaPIs; ultimately derived from ancestral phages or interference. PtiA binds to the phage 80 late gene transcription activator protophages, the SaPIs have undergone an evolutionary di- protein, LtrC, and blocks LtrC-mediated phage late gene activation, which is vergence that has included the acquisition of a special category essential for the expression of packaging and lysis genes. A modulator of phage-resistance mechanisms not used by other genetic ele- protein, PtiM, binds to and precisely modulates the activity of PtiA, attaining ments. These mechanisms differ from the usual phage-resistance the required level of interference. Preliminary data show that another SaPI2 mechanisms in that they are carefully crafted to ensure the protein, PtiB, also inhibits the phage late gene transcription, but its mech- production of mature phage particles, most of which contain anism remains to be elucidated. SaPI rather than phage DNA. Like other phage-resistance mechanisms, the SaPIs target essential phage functions, and all limiting for SaPI particle production and that this limitation is known thus far affect particle maturation and DNA packaging. not overcome by increasing the availability of virion proteins. The two SaPI resistance mechanisms first identified target helper This limiting factor could only be TerS or the SaPI-specific phage capsid morphogenesis and DNA packaging, respectively, S functions that do not compromise SaPI particle production. The replication initiator protein. Because there always is excess SaPI third mechanism, described here, is unique in that it involves two DNA at the time of cellular lysis, the latter possibility could be ruled out. proteins, PtiA and PtiB, that target the promoter of late phage ptiA ptiB gene transcription, which is responsible for the production of However, once it was determined that both and in- hibit transcription of the bacteriophage late gene promoter (Fig. virion and lysis proteins. This interference mechanism must be B modulated precisely so as not to interfere with SaPI particle 5 ), which is responsible for production of virion proteins, the formation, and we show here that a modulatory protein, PtiM, expectation would be that phage and SaPI titers would be af- binds to one of the interference-mediating proteins, PtiA. The fected equally, because the reduction in the overall level of virion other protein, PtiB probably is modulated also, but, because its proteins should take precedence over the putative rate limitation extreme toxicity for the host cell, it has not yet been analyzed. of TerSS. This expectation was not realized: As shown in Table ptiA ptiA ptiB Thus, as shown in the diagram in Fig. 6, PtiA has two binding S1, deletion of either S2 alone or of S2 and S2 had no partners, one that it inhibits and one that inhibits it. This in- significant effect on SaPI titer, although phage titer was in- terference mechanism, like the others, favors the SaPI over its creased significantly. Given that the morphogenetic (late) phage helper phage, and one would assume that phage and SaPI are in proteins generally are present in considerable excess, the effect competition for particle maturation so that deletion of the in- of this rather modest inhibition of the Ltr promoter (Fig. 5B) terference genes would result in an increase in phage titer con- suggests that production of one or more late phage proteins, comitantly with diminished production of SaPI particles. In such as TerSP, is rate-limiting for the phage and that this rate reality, the situation is rather more complex. In a previous study limitation would be overcome by relief of LtrC inhibition. The (7), we observed that deletion of an interference gene increased biological basis for interference by SaPI with late phage tran- phage titer but did not decrease SaPI titer. The biological scription would be precisely parallel to that suggested for the function of SaPI-mediated interference was, as expected, to previously described ppi gene (7), despite the nature of the target diminish phage particle production relative to SaPI particle of inhibition—namely, to give the SaPI an advantage over its production. The increase in phage titer upon deletion of an helper phage. Because the inhibited protein is required for both interference gene is consistent with the well-known excess of SaPI and phage particle production, it must be modulated pre- virion proteins in the infected cell. To explain the failure of SaPI cisely to ensure adequate production of both types of particles. particle production to respond to the presence or absence of the Note, however, that, as shown in Table S1, deletion of ptiBS2 , interference genes, we suggest that a SaPI product must be rate- unlike the deletion of ptiAS2, did cause a modest reduction in

Ram et al. PNAS Early Edition | 5of6 Downloaded by guest on September 24, 2021 SaPI titer. To explain this effect, we hypothesize that, in addition conditions are described in SI Materials and Methods. Staphylococcal tem- to its inhibitory role, PtiB may have a regulatory role, such as up- perate phages 80, 80α, 52A, 71, ETA, and ϕNM4 were used in this study. regulation of operon 1 transcription, which would increase TerSS production. Why deletion of both genes reverses this effect is DNA Methods. All general DNA manipulations (e.g., digestion and ligation) unexplained. The location of terSS in the same operon as two of were carried out by standard methods (23). Oligonucleotides used for this the three interference genes (Fig. 1B) raises an interesting reg- work are listed in Table S4. All restriction enzymes and T4 DNA ligase were purchased from New England Biolabs. Primers were obtained from Macro- ulatory question: terSS surely would be up-regulated during SaPI induction, certainly would be up-regulated by relief of LexA gen. Integrated DNA Technologies Inc. performed DNA sequencing. repression of operon 1, and perhaps also would be up-regulated by the up-regulation of one or more promoters upstream of Mutant Construction. Mutants (in-frame deletion) were generated using the operon, which would be needed for the transcription of ppi allelic exchange vector pMAD (24) as previously described (18). Details are (ORF10), which is immediately upstream of operon 1 (18). The given in SI Materials and Methods. other interference genes would be up-regulated also, leading to Phage Infection and Induction of Lysogen. S. aureus strains were inoculated in diminution of phage particle production, as observed. However, casamino acids yeast extract glycerophosphate (CYGP) (25) broth at an initial because TerS , like other TerS’s, is presumed to be a multi- S OD = 0.1 (∼1 × 108 cfu/mL) and were grown at 37 °C and 225 rpm. For subunit enzyme, an increase in the TerS holoenzyme would 600 phage infection, cultures were adjusted to OD = 1.0 (∼1 × 109 cfu/mL) necessarily be accompanied by a much greater increase in the 600 with CYGP broth and diluted 1:1 with phage buffer (25) infected with phage interference protein copy numbers. The effects of this increase 80. To induce expression during phage growth of genes cloned in plasmid would necessarily be mitigated by PtiM for PtiA and by a puta- pCN51, 1.0 μM of CdCl was added at the time of infection. For lysogen tive, as yet unknown, modulator for PtiB. Experiments to sort 2 induction, cultures were adjusted to OD = 1.0 (∼1 × 109 cfu/mL) with CYGP out this interesting regulatory circuitry are in progress. 600 broth and diluted 1:1 with CYGP broth containing mitomycin C (final con- We and others (19) predict that since the SaPIs package their centration, 2 μg/mL). DNA the same way that phages do, the SaPIs should be able to pac mediate generalized , using site homologs in Plaque Assay. Approximately 100–200 phage 80 particles were mixed with host DNA that would be recognized by TerS . By interfering with 8 9 S ∼10 cells (100 μL culture adjusted to OD600 = 1.0; ∼1 × 10 cfu/mL) of helper phage reproduction, the SaPIs would thus promote hor- RN4220 and its derivative strains. This mixture was incubated at room izontal transfer of a wide variety of host genes in addition to temperature for 15 min, subsequently mixed with 3 mL of phage top agar their own transfer. It is interesting to compare this mechanism (25), and immediately poured on a phage bottom agar (25) plate without with an entirely different phage-interference mechanism, that of or with 1.0 μM CdCl2. Plates were incubated at 32 °C for 48 h and stained the clustered regularly interspaced short palindromic repeats with 0.1% triphenyl tetrazolium chloride (TTC) (Difco) (26) in tryptic soy (CRISPRs) (20, 21), which block phage propagation completely broth (TSB). by degrading incoming phage DNA and thus block all phage- mediated (22). Because the SaPIs are Enzyme Assays. Phage late gene transcription was measured using the extremely common, with most S. aureus strains harboring at least β-lactamase enzyme assay using nitrocefin as substrate. The assays were one, whereas the CRISPRs are extremely rare in S. aureus,itcould performed as mentioned (27) using a Thermomax (Molecular Devices)

be argued that the SaPIs have won that particular evolutionary microtiter plate reader. β-Lactamase units are defined as Vmax/OD650. Details of contest. All-in-all, the SaPI-phage interactions represent a remark- preparation of samples for this assay are described in SI Materials and Methods. able microcosm within the bacterial intracellular universe. Bacterial two-hybrid assays, the one-step growth curve, and PtiB toxicity assays are described in SI Materials and Methods. Materials and Methods Bacterial Strains and Growth Conditions. Bacterial strains and plasmids used in ACKNOWLEDGMENTS. This work was supported by National Institutes of these studies are listed in Tables S2 and S3, respectively. Growth media and Health Grant R01AI022159 (to R.P.N.).

1. Novick RP, Christie GE, Penadés JR (2010) The phage-related chromosomal islands of 14. Grayhack EJ, Roberts JW (1982) The phage lambda Q gene product: Activity of Gram-positive bacteria. Nat Rev Microbiol 8(8):541–551. a transcription antiterminator in vitro. Cell 30(2):637–648. 2. Tormo-Más MA, et al. (2010) Moonlighting bacteriophage proteins derepress staph- 15. Deighan P, Hochschild A (2007) The bacteriophage lambdaQ anti-terminator protein ylococcal pathogenicity islands. Nature 465(7299):779–782. regulates late gene expression as a stable component of the transcription elongation 3. Ruzin A, Lindsay J, Novick RP (2001) Molecular genetics of SaPI1—a mobile patho- complex. Mol Microbiol 63(3):911–920. genicity island in Staphylococcus aureus. Mol Microbiol 41(2):365–377. 16. Ubeda C, et al. (2007) SaPI operon I is required for SaPI packaging and is controlled 4. Tallent SM, Langston TB, Moran RG, Christie GE (2007) Transducing particles of by LexA. Mol Microbiol 65(1):41–50. Staphylococcus aureus pathogenicity island SaPI1 are comprised of helper phage- 17. S. L (2012) Lactic Acid Bacteria: Microbiological and Functional Aspects, Chapter 9, – encoded proteins. J Bacteriol 189(20):7520 7524. (CRC, Boca Raton, FL), 4th Ed. 5. Tormo MA, et al. (2008) Staphylococcus aureus pathogenicity island DNA is packaged 18. Ubeda C, et al. (2008) SaPI mutations affecting replication and transfer and enabling in particles composed of phage proteins. J Bacteriol 190(7):2434–2440. autonomous replication in the absence of helper phage. Mol Microbiol 67(3):493–503. 6. Ubeda C, et al. (2009) Specificity of staphylococcal phage and SaPI DNA packaging as 19. Bento JC, et al. (2014) Sequence determinants for DNA packaging specificity in the revealed by integrase and terminase mutations. Mol Microbiol 72(1):98–108. S. aureus pathogenicity island SaPI1. Plasmid 71:8–15. 7. Ram G, et al. (2012) Staphylococcal pathogenicity island interference with helper 20. Barrangou R, et al. (2007) CRISPR provides acquired resistance against viruses in phage reproduction is a paradigm of molecular parasitism. Proc Natl Acad Sci USA prokaryotes. Science 315(5819):1709–1712. 109(40):16300–16305. 21. Deveau H, Garneau JE, Moineau S (2010) CRISPR/Cas system and its role in phage- 8. DeLeo FR, et al. (2011) Molecular differentiation of historic phage-type 80/81 and bacteria interactions. Annu Rev Microbiol 64:475–493. contemporary epidemic Staphylococcus aureus. Proc Natl Acad Sci USA 108(44): 22. Marraffini LA, Sontheimer EJ (2008) CRISPR interference limits horizontal gene 18091–18096. – 9. Charpentier E, et al. (2004) Novel cassette-based shuttle vector system for gram- transfer in staphylococci by targeting DNA. Science 322(5909):1843 1845. positive bacteria. Appl Environ Microbiol 70(10):6076–6085. 23. Sambrook JRD (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor 10. Corbisier P, Ji G, Nuyts G, Mergeay M, Silver S (1993) luxAB gene fusions with the Laboratory Press, Cold Spring Harbor, NY). arsenic and cadmium resistance operons of Staphylococcus aureus plasmid pI258. 24. Arnaud M, Chastanet A, Débarbouillé M (2004) New vector for efficient allelic re- FEMS Microbiol Lett 110(2):231–238. placement in naturally nontransformable, low-GC-content, gram-positive bacteria. 11. Ferrer MD, et al. (2011) RinA controls phage-mediated packaging and transfer of Appl Environ Microbiol 70(11):6887–6891. virulence genes in Gram-positive bacteria. Nucleic Acids Res 39(14):5866–5878. 25. Novick RP (1991) Genetic systems in staphylococci. Methods Enzymol 204:587–636. 12. Quiles-Puchalt N, et al. (2013) A super-family of transcriptional activators regulates bacte- 26. Pattee PA (1966) Use of tetrazolium for improved resolution of bacteriophage plaques. riophage packaging and lysis in Gram-positive bacteria. Nucleic Acids Res 41(15):7260–7275. JBacteriol92(3):787–788. 13. Christie GE, et al. (2010) The complete genomes of Staphylococcus aureus bacteriophages 27. Ji G, Beavis R, Novick RP (1997) Bacterial interference caused by autoinducing 80 and 80α—implications for the specificity of SaPI mobilization. Virology 407(2):381–390. peptide variants. Science 276(5321):2027–2030.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1406749111 Ram et al. Downloaded by guest on September 24, 2021