Antirepression system associated with the life cycle switch in the temperate podoviridae phage SPC32H

Abstract

Prophages switch from lysogenic to lytic mode in response to the host SOS response. The primary factor that governs this switch is a phage repressor, which is typically a host RecA-dependent autocleavable protein. Here, in an effort to reveal the mechanism underlying the phenotypic differences between the Salmonella temperate phages SPC32H and SPC32N, whose genome sequences differ by only two nucleotides, we identified a new class of Podoviridae phage lytic switch antirepressor that is structurally distinct from the previously reported Sipho- and Myoviridae phage antirepressors. The SPC32H repressor (Rep) is not cleaved by the SOS response but instead is inactivated by a small antirepressor (Ant), the expression of which is negatively controlled by host LexA. A single nucleotide mutation in the consensus sequence of the LexA-binding site, which overlaps with the ant promoter, results in constitutive Ant synthesis and consequently induces SPC32N to enter the lytic cycle. Numerous potential Ant homologues were identified in a variety of putative prophages and temperate Podoviridae phages, indicating that antirepressors may be widespread among temperate phages in the order Caudovirales to mediate a prudent prophage induction.

Fig 1

Two similar S. Typhimurium-specific Podoviridae phages, SPC32H and SPC32N, produce morphologically distinct plaques. (A and B) Plaque morphology of SPC32H (A) or SPC32N (B). Dilutions (10 μl) of each phage stock were spotted onto a lawn of the S. Typhimurium LT2(c) Δ^LT2gtrABC1 strain (SR5003). (C and D) TEM image of SPC32H (C) or SPC32N (D). Inset at the bottom left of each panel shows the enlarged virion morphology with a black scale bar (50 nm). The white arrow and arrowheads indicate the tail shaft and tail spikes, respectively.

Fig 2

There are two single nucleotide differences between the genomes of ε15-like phage SPC32H and SPC32N. (A) DNA alignment of the genomes of phage ε15 (NC_004775.1), SPC32H, and phiV10 (NC_007804.2) using Easyfig. High sequence similarity between the genomes is indicated by the gray regions. SPC32H ORFs are indicated by numbered or annotated arrows. Phage functional modules are indicated under the arrows. ant, antirepressor; tsp, tailspike; oac, o-acetyltransferase; hol, holin; end, endolysin; int, integrase; rep, repressor. Note that the SPC32N genome is identical to that of SPC32H with the exception of two single nucleotide differences (see panel B). (B) Schematic representation of the location of the two single nucleotide differences, m1 and m2. The partial SPC32H genome sequence surrounding the two single nucleotide differences is shown. m1 (located within the tsp gene) and m2 (located in the intergenic region between SPC32H_020 and tsp) are indicated in bold, uppercase letters. The predicted −10 and −35 sites of the putative promoter for SPC32H_020 gene are boxed. The putative LexA-binding site (SOS box) and the putative repressor-binding site are underlined and doubly underlined, respectively. (C) Consensus sequence of the LexA-binding site from E. coli (8, 33, 34) and the putative LexA-binding sites from phage SPC32H and SPC32N. m2 is indicated with a gray background. Note that the LexA-binding site sequences for SPC32H and SPC32N shown here are reverse complements of the sequence shown in panel A.

Fig 3

Introducing the m2 sequence from SPC32N induces SPC32H to enter the lytic cycle. (A) High-titer phage stocks (>10⁷ PFU ml⁻¹; 10 μl) of SPC32H, SPC32N, and three mutant phages derived from SPC32H were spotted onto a lawn of S. Typhimurium LT2(c) Δ^LT2gtrABC1. (B) SPC32H can lysogenize host Salmonella, whereas SPC32N cannot. Various template samples were PCR amplified with an attR-specific primer pair. M, DNA marker 1 Kb⁺ (Invitrogen); i, inner part of the lysis zone; e, edge of the lysis zone; gDNA, genomic DNA; Δ^LT2gtrABC1(32H), SPC32H lysogen (SR5100). (C) DNA isolated from the lysis zones shown in panel A was PCR amplified using primers specific for the attR site to determine the lysogenization of each phage. Lanes 1 to 5 correspond to each lysis zone shown in panel A. Note that the introduction of m2 resulted in a disappearance of the lysogen-specific attR band (lanes 4 and 5).

Fig 4

The novel antirepressor, encoded by SPC32H_020 (ant), induces the lytic development of SPC32H. (A) Supplementation with the putative repressor leads to the lysogenic development of the lytic cycle-biased phage SPC32N, while supplementation with the putative antirepressor results in the lytic development of SPC32H. Salmonella strains transformed with a control plasmid (pBAD24), a putative repressor-overexpressing plasmid (prep), or an SPC32H_020-overexpressing plasmid (pant) were infected with serially diluted (10-fold) stocks of SPC32H or SPC32N. l-Arabinose (0.2%, final concentration) was added to induce SPC32H_020 expression from pant. (B) The expression of the SPC32H_020 protein promotes the switch from lysogenic to lytic development. The SPC32H lysogen [Δ^LT2gtrABC1 (32H); SR5100] and nonlysogen (Δ^LT2gtrABC1; SR5003) strains were transformed with pant or a control plasmid (pBAD24), and the resulting strains were subjected to a disc diffusion assay with 10 μl of 15% l-arabinose. pant* indicates the plasmid encoding a frame-shifted ant gene. Arabinose-induced bacterial lysis was observed only in the SPC32H lysogen harboring pant.

Fig 5

The ant promoter of SPC32H is activated by DNA damage via LexA proteolysis, whereas the SPC32N ant promoter is constitutively active, due to the inability of LexA to bind to the m2-containing consensus LexA-binding site. The RLU (relative light units) were calculated by dividing the measured bioluminescence by the A₆₀₀ value. The mean and SD for three independent assays are shown on a log scale on the y axis (A and B). (A) Time course observation of ant promoter activity in the presence or absence of DNA damage. Salmonella strains harboring the bioluminescence reporter plasmid pP_{ant_H}::lux (luxCDABE fused to the putative ant promoter of SPC32H) or pP_{ant_N}::lux (luxCDABE fused to the putative ant promoter of SPC32N) were incubated at 37°C, and the bioluminescence, as well as the A₆₀₀ of the culture, was measured every half-hour. The vertical arrows indicate MMC treatment (1 μg ml⁻¹, final concentration; 3 h after incubation). (B) ant promoter activities of the various Salmonella strains at an A₆₀₀ of ∼0.6, harboring the bioluminescence plasmid. MMC (1 μg ml⁻¹, final concentration) was added after 3 h of incubation. lexA⁺, Δ^LT2gtrABC1, SR5003; lexA⁺(32H), Δ^LT2gtrABC1(32H), SR5100; ΔlexA, Δ^LT2gtrABC1 ΔsulA ΔlexA, SR5158; lexA(G85D), Δ^LT2gtrABC1 ΔsulA lexA(G85D), SR5176. ∗∗∗, P < 0.001. (C) LexA specifically binds to the putative ant gene promoter region of SPC32H but not to that of SPC32N, which contains m2. The γ-³²P-labeled DNA fragment of the ant gene promoter region from SPC32H (APR_H*) or from SPC32N (APR_N*) was incubated with the indicated amounts of purified Salmonella LexA and was subjected to an electrophoretic mobility shift assay (EMSA). Corresponding unlabeled DNA fragments (APR_H and APR_N) were used for the competition analysis. The position of the unbound fragments (F) and fragments retarded by LexA binding (B) are indicated.

Fig 6

DNA damage-induced LexA proteolysis followed by SPC32H ant expression induces the switch to lytic development. The lacZ gene, transcriptionally fused to the putative recET genes, was introduced into the SPC32H lysogens harboring an intact (lexA⁺) or noncleavable [lexA(G85D)] LexA, and the resulting strains were subjected to a disc diffusion assay with the following solutions: MMC, 0.5 mg ml⁻¹ mitomycin C; Cm, 2.5 mg ml⁻¹ chloramphenicol; Ap, 10 mg ml⁻¹ ampicillin; D.W., distilled water. Note that the blue zone appears to surround the MMC disc in the lexA⁺ background only.

Fig 7

DNA damage induces Ant accumulation but not Rep cleavage, and the consequent binding of Ant to Rep inhibits the binding of Rep to specific operators. (A) Salmonella strains lysogenized by SPC32H expressing HA-tagged Rep (upper panel; Δ^LT2gtrABC1 [32H rep-HA], SR5192) or both HA-tagged Rep and HA-tagged Ant (lower panel; Δ^LT2gtrABC1 [32H rep-HA ant-HA], SR5197) were exposed to MMC for 1 or 2 h, respectively. The MMC-treated bacterial cultures were sampled at the indicated time points and subjected to the Western blotting to immunodetect the HA-tagged proteins. DnaK was used as an internal control. (B) Bacterial two-hybrid assays revealed the direct binding of Ant to Rep. The β-galactosidase activity of E. coli BTH101 reporter strains harboring the indicated plasmid pairs were measured. The activities are presented in Miller units. B, a backbone plasmid. (C) EMSA with purified Rep and Ant demonstrates the Ant-mediated inhibition of Rep binding to its operators. Mixtures of APR_H* and the indicated amounts of Rep were incubated at 20°C for 15 min in 1× binding buffer supplemented with 1.1 μg of poly(dI-dC) and then electrophoresed on a 6% native acrylamide slab gel for EMSA. For competition analysis, unlabeled APR_H fragments were added as cold probes to the mixture. When appropriate, Rep was preincubated with the indicated amounts of Ant at 20°C for 30 min and further incubated with APR_H* as described above. The positions of the unbound fragments (F) and fragments retarded by Rep binding (B1 and B2) are indicated.

Fig 8

Amino acid alignment of the phage antirepressors. The amino acid sequences of Tum (from coliphage 186), AntC (from coliphage N15), GfoA (from Gifsy-1) and Ant (from SPC32H) were aligned using ClustalW2. There are no noticeable consensus residues, demonstrating the diversity of phage antirepressors in the order Caudovirales.