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, 111 (40), 14536-41

Precisely Modulated Pathogenicity Island Interference With Late Phage Gene Transcription

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Precisely Modulated Pathogenicity Island Interference With Late Phage Gene Transcription

Geeta Ram et al. Proc Natl Acad Sci U S A.

Abstract

Having gone to great evolutionary lengths to develop resistance to bacteriophages, bacteria have come up with resistance mechanisms directed at every aspect of the bacteriophage life cycle. Most genes involved in phage resistance are carried by plasmids and other mobile genetic elements, including bacteriophages and their relatives. A very special case of phage resistance is exhibited by the highly mobile phage satellites, staphylococcal pathogenicity islands (SaPIs), which carry and disseminate superantigen and other virulence genes. Unlike the usual phage-resistance mechanisms, the SaPI-encoded interference mechanisms are carefully crafted to ensure that a phage-infected, SaPI-containing cell will lyse, releasing the requisite crop of SaPI particles as well as a greatly diminished crop of phage particles. Previously described SaPI interference genes target phage functions that are not required for SaPI particle production and release. Here we describe a SaPI-mediated interference system that affects expression of late phage gene transcription and consequently is required for SaPI and phage. Although when cloned separately, a single SaPI gene totally blocks phage production, its activity in situ is modulated accurately by a second gene, achieving the required level of interference. The advantage for the host bacteria is that the SaPIs curb excessive phage growth while enhancing their gene transfer activity. This activity is in contrast to that of the clustered regularly interspaced short palindromic repeats (CRISPRs), which totally block phage growth at the cost of phage-mediated gene transfer. In staphylococci the SaPI strategy seems to have prevailed during evolution: The great majority of Staphylococcus aureus strains carry one or more SaPIs, whereas CRISPRs are extremely rare.

Keywords: bacteriophage resistance; helper phage; transcription regulation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
(A) Phage interference mediated by cloned SaPI2 genes. The indicated genes were cloned to plasmid pCN51 under a cadmium-inducible promoter (Pcad). Strain RN4220 containing the indicated plasmids was infected with phages 80α (∼150 pfu per plate) or 80 (∼250 pfu per plate) plated on phage bottom agar containing 1.0 μM CdCl2 and incubated for 48 h at 32 °C. Plates were stained with 0.1% TTC in TSB and photographed. (B) Map of SaPI2 operon 1. Genes previously shown to cause phage interference are shown in red; hypothetical genes of unknown function are shown in blue; numbered ORFs newly determined to be involved in interference are shown in orange; and the SaPI2 small terminase subunit is shown in green.
Fig. 2.
Fig. 2.
(A) Comparison of the rinA and ltrC genomic region from 80α and 80. Note that the position of ltrC (ORF32) in phage 80 corresponds to that of rinA in phage 80α. “P” represents the late gene promoter. (B) Activation of phage 52A late transcript promoter by LtrC. The late transcript promoter was cloned to plasmid pCN41 as a transcriptional fusion to β-lactamase. The resulting reporter plasmid was introduced into strains lysogenic for either the WT prophage or a derivative prophage containing a deletion of ltrC in the absence or presence of SaPI2. β-Lactamase activity was measured 2 h after induction with mitomycin C (MC) and in a parallel culture without induction. (C) Effects of cloned ptiA and ptiM on the phage late transcript promoter. ptiA and ptiM were cloned singly or together to pCN51, and their effects on LtrC activation of the late transcript promoter–β-lactamase fusion were determined. For these tests, the pti genes were induced with 1.0 µm CdCl2, the β-lactamase fusion construct was integrated in the chromosome at the SaPI4 att site (lab plasmids collection), and LtrC was provided by superinfection with phage 80. Cultures were assayed at 2 h postinfection, and assays were performed in triplicate.
Fig. 3.
Fig. 3.
(A) Modulation of PitA activity by PtiM. (Upper) Plaque assay. Phage platings were as in Fig. 1A. (Lower) Lysis assay. RN4220strains carrying pCN51 (vector) or carrying the pti clones used were grown with the inducer (1.0 μM CdCl2), infected with phage 80 at a multiplicity of infection (MOI) of 0.2, and incubated at 32 °C at 60 rpm for 3 h. (B) Bacterial adenylate cyclase-based two-hybrid (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-LtrC (WT80) + pUT18-PtiAS2; 3, pKT25-LtrC (PtiAS2-resistant 80) + pUT18-PtiAS2; 4, pKT25-PtiMS2 + pUT18-PtiAS2; 5, pKT25 + pUT18 (negative control). Blue color indicates cAMP-dependent lacZ expression following reconstitution of adenylate cyclase activity by the interaction of fusion proteins.
Fig. 4.
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 incubated for 48 h at 32 °C. Plates were stained with 0.1% TTC in TSB and photographed.
Fig. 5.
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.
Fig. 6.
Fig. 6.
Summary of SaPI2-mediated interference with phage 80. SaPI2 operon 1 encodes three proteins, PtiA, PtiM, and PtiB, involved in phage 80 interference. PtiA binds to the phage 80 late gene transcription activator protein, LtrC, and blocks LtrC-mediated phage late gene activation, which is essential for the expression of packaging and lysis genes. A modulator protein, PtiM, binds to and precisely modulates the activity of PtiA, attaining the required level of interference. Preliminary data show that another SaPI2 protein, PtiB, also inhibits the phage late gene transcription, but its mechanism remains to be elucidated.

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