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Precisely Modulated Pathogenicity Island Interference with Late Phage Gene Transcription

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Precisely Modulated Pathogenicity Island Interference with Late Phage Gene Transcription Precisely modulated pathogenicity island 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 bacteriophages, bacteria have come up with resistance mech- reproduction of their helper phages (7). Thus far, two distinctly anisms directed at every aspect of the bacteriophage 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 mobile genetic elements, 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 genome, 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- genomes 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 (CRISPRs), 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 Staphylococcus aureus 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 prophage 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.
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