anti-repression system associated with the life cycle...

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Anti-Repression System Associated with the Life Cycle Switch in the 4"

Temperate Podoviridae Phage SPC32H 5"

6"

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Minsik Kim and Sangryeol Ryu# 8"

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Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, Research :"

Institute for Agriculture and Life Sciences, and Center for Food and Bioconvergence, Seoul National ;"

University, Seoul 151-921, South Korea 32"

33"

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Correspondence to: Sangryeol Ryu, Department of Food and Animal Biotechnology, Seoul National 35"

University, Seoul 151-921, Korea. Tel.: +82 2 880 4856; Fax: +82 2 873 5095; E-mail: 36"

[email protected] 37"

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Running title: Anti-Repression System in a Lytic Switch of Podophage 3:"

3;"

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Keywords: bacteriophage/ anti-repressor/ lytic switch/ SOS response / i15-like phage 43"

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The word count for the abstract: 182 words 47"

The word count for the text: 5430 words 48"

JVI Accepts, published online ahead of print on 28 August 2013J. Virol. doi:10.1128/JVI.02173-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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Abstract 49"

Prophages switch from lysogenic to lytic mode in response to the host SOS response. The 4:"

primary factor that governs this switch is a phage repressor, which is typically a host RecA-dependent 4;"

autocleavable protein. Here, in an effort to reveal the mechanism underlying the phenotypic 52"

differences between the Salmonella temperate phages SPC32H and SPC32N, whose genome 53"

sequences differ by only two nucleotides, we identified a new class of Podoviridae phage lytic switch 54"

anti-repressor that is structurally distinct from the previously reported Sipho- and Myoviridae phage 55"

anti-repressors. The SPC32H repressor (Rep) is not cleaved by the SOS response but, instead, is 56"

inactivated by a small anti-repressor (Ant), the expression of which is negatively controlled by host 57"

LexA. A single nucleotide mutation in the consensus sequence of the LexA-binding site, which 58"

overlaps with the ant promoter, results in constitutive Ant synthesis and, consequently, induces 59"

SPC32N to enter the lytic cycle. Numerous potential Ant homologues were identified in a variety of 5:"

putative prophages and temperate Podoviridae phages, indicating that anti-repressors may be 5;"

widespread among temperate phages in the order Caudovirales to mediate a prudent prophage 62"

induction. 63"

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Introduction 65"

Bacteriophages (phages), which are natural viral predators of bacteria, multiply by infecting 66"

specific host bacteria. Although there is an additional type of phage-host relationship called “steady-67"

state infection”, which is exemplified by filamentous phages (1), phage genome replication generally 68"

occurs via two different developmental paths: the lytic cycle and the lysogenic cycle. In contrast to the 69"

lytic cycle, which results in immediate bursting of the host bacteria and the release of bacteriophage 6:"

progeny, the lysogenic cycle involves the maintenance of the phage genome as a part of the host 6;"

genome for several generations, typically by integrating into host chromosomes or, more rarely, by 72"

replicating as low-copy-number phage-plasmids"(2-4). The expression of genes necessary for progeny 73"

production and host cell lysis are tightly repressed by a phage regulatory system, but some 74"

physiological changes in the host induced by UV light irradiation or other DNA-damaging agents 75"

activate the lytic cycle by disabling the phage repressor. Phages fall into two categories: virulent 76"

phages that replicate strictly by the lytic cycle and temperate phages that can enter both the lytic cycle 77"

and the lysogenic cycle. 78"

The lytic switch following lysogenic development has been well-studied in the temperate phage 79"

lambda. In the lambda lysogenic phase, phage CI repressors form dimers and bind to specific 7:"

operators to prevent expression of lambda early genes and subsequent late genes"(5, 6). Upon host 7;"

DNA damage, the activated host RecA protein induces CI proteolysis in a manner similar to the 82"

inactivation of the host SOS response regulator LexA (7-9). CI proteolysis leads to the expression of 83"

early and late genes, resulting in lytic development. This mechanism illustrates how lambda and other 84"

similar phages exploit the host cell SOS response to escape quickly from a potentially damaged host 85"

using the RecA-dependent cleavable repressor. Alternatively, some phages in the family Sipho- and 86"

Myoviridae utilize the LexA-regulated anti-repressors instead of the cleavable repressor to associate 87"

their lytic switch to the host SOS response (10-12). 88"

Here in an effort to identify the factor(s) that causes a phenotypic difference between two very 89"

similar podoviral Salmonella phages, SPC32H and SPC32N, we found a novel Podoviridae phage 8:"

lytic switch anti-repressor. We observed that a single nucleotide change in the LexA-binding site, 8;"


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which overlaps with the promoter of phage anti-repressor gene, causes constitutive expression of the 92"

anti-repressor Ant and consequent inhibition of phage repressor function in SPC32N. As a result, 93"

SPC32N could not establish lysogeny as clear plaque mutants. A LexA-dependent lytic switch 94"

involving an anti-repressor, rather than repressor proteolysis, has been reported previously in only 95"

siphon- and myoviral phages (10-12), and the podoviral SPC32H/N Ant protein had no significant 96"

homology to these known anti-repressors. A database search identified many proteins with homology 97"

to Ant, suggesting the extensive use of anti-repressor-mediated lytic induction among temperate 98"

phages in the order Caudovirales. 99"

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Materials and Methods 9;"

:2"

Bacterial strains, plasmids, and growth conditions :3"

All Salmonella mutants were derived from the prophage-cured S. Typhimurium strain LT2 :4"

(referred to as LT2(c)) and its derivative 〉LT2gtrABC1 (SR5003) to exclude the effect of prophages :5"

and spontaneous phage-resistance via O-antigen glucosylation, respectively (13, 14). Standard cloning :6"

procedures were used to construct the recombinant plasmids. Bacteria were grown aerobically at 37°C :7"

in LB medium supplemented with the following chemicals, as needed: ampicillin (Ap), 50 たg ml-1; :8"

kanamycin (Km), 50 たg ml-1

; chloramphenicol (Cm), 25 たg ml-1

; 5-bromo-4-chloro-3-indolyl-く-D-:9"

galactoside (X-gal), 40 たg ml-1; L-arabinose, 0.2% (final concentration); isopropyl-く-D-::"

thiogalactopyranoside (IPTG), 1000 たM (final concentration); and mitomycin C (MMC), 1 たg ml-1 :;"

(final concentration). For the disc diffusion assay, 6-mm-diameter filter paper discs were soaked with ;2"

10 たl of arabinose, antibiotics or MMC at the indicated concentrations, placed on the surface of the ;3"

bacteria-inoculated solidified soft top agar (LB supplemented with 0.4% (wt/vol) agar and X-gal, if ;4"

necessary), and incubated at 37°C for 8 hr. ;5"

;6"

Bacteriophage ;7"

The bacteriophages used in this study are listed in Table 1. All phage mutants were derived from ;8"

the temperate phage SPC32H, which was previously isolated from chicken fecal samples obtained ;9"

from a traditional marketplace in South Korea (15). Routine phage spotting and double-agar overlay ;:"

assays were conducted to determine the efficiency of plating (EOP) in specific bacteria (14, 15). For ;;"

the morphological analysis, the phage stocks were negatively stained with 2% uranyl acetate (pH 4.0) 322"

as previously described (15) and were examined by transmission electron microscopy (LEO 912AB 323"

TEM; Carl Zeiss, Jena, Germany) at 120-kV accelerating voltage. The images were scanned with a 324"

Proscane 1,024 X 1,024-pixel charge-coupled device camera. 325"

326"

Bacteriophage genome sequencing and analysis 327"


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Phage nucleic acids that were extracted by the phenol/chloroform extraction method with 328"

protease K/SDS treatment" (16) were pyrosequenced using GS FLX Titanium by Macrogen, Seoul, 329"

South Korea. The quality-filtered reads were assembled using GS De Novo assembler (v. 2.60), and 32:"

the open reading frames (ORFs) that encode proteins more than 35 amino acids were predicted using 32;"

GeneMarkS" (17), Glimmer 3.02" (18) and FgenesB (Softberry, Inc., Mount Kisco, NY, USA). The 332"

predicted ORFs were annotated based on the results of BlastP" (19), InterProScan" (20) and NCBI 333"

Conserved Domain Database" (21) analysis. tRNAscan-SE" (22) and BPROM (Softberry, Inc.) were 334"

used to predict the tRNA sequences and the putative promoter/transcription factor-binding sites, 335"

respectively. Genomic comparison at the DNA level was visualized using Easyfig (23). 336"

337"

Construction of the Salmonella and phage mutants 338"

The lambda Red recombination method was used for in-frame gene deletion (24). To construct 339"

the non-cleavable LexA, a point mutation in lexA (resulting in a G85D mutation in the amino acid 33:"

sequence) was generated by lambda Red recombination and double homologous recombination-based 33;"

counter-selection, as previously described (14), using the suicide vector pDS132 (25). The SPC32H 342"

lysogen [〉LT2gtrABC1 (32H); SR5100] was isolated by sequential streaking of SPC32H-resistant 343"

clones from a lawn of phage-treated 〉LT2gtrABC1 and was verified by PCR amplification of the phage 344"

attachment (attR) site. The transcriptional recET::lacZ fusion was constructed using pCE70," as 345"

previously described (26, 27). Human influenza hemagglutinin (HA) epitope tagging of specific 346"

gene(s) was also accomplished by lambda Red recombination using oligonucleotides containing the 347"

HA-tag sequence. 348"

Phage mutants were induced from the SPC32H lysogen after the gene manipulations described 349"

above, with some modifications. Briefly, to generate SPC32H m1, a truncated tailspike gene 34:"

(tsp::Kmr), which was constructed by lambda Red recombination in the SPC32H lysogen, was 34;"

replaced with the m1-containing tsp gene by double homologous recombination-based counter-352"

selection. The presence of the m1 mutation in the induced phage was confirmed by DNA sequencing. 353"

Similar methods were used to construct SPC32H m2 and SPC32H m12. The oligonucleotides used in 354"


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this study are listed in Table 3. 355"

356"

Bioluminescence reporter assay 357"

The 197-bp fragment upstream of the ant gene in SPC32H (designated Pant_H) or SPC32N 358"

(designated Pant_N) was PCR amplified and cloned into the pBBRlux (28), resulting in the 359"

transcriptional fusion of the operon luxCDABE to the putative ant gene promoter. S. Typhimurium 35:"

strains harboring this reporter plasmid were cultured in 200-たl of fresh LB broth supplemented with 35;"

appropriate antibiotics in a 96-well plate. The cellular bioluminescence of the culture as well as the 362"

absorbance at 600 nm (A600) was measured periodically using an Infinite® 200 PRO (Tecan, 363"

Männedorf, Switzerland), and the results were expressed in arbitrary relative light units (RLU). To 364"

trigger the SOS responses, MMC was added to the culture after 3-hr incubation. The three 365"

independent assays with triple technical replications were performed. 366"

367"

Western blot analysis 368"

At the mid-exponential phase, culture of the HA-tagged gene(s)-containing Salmonella was 369"

treated by MMC, and portions of the culture were sampled at the indicated time points. Bacterial cells 36:"

were harvested by centrifugation, and were lysed with B-PER reagent (Thermo Scientific, IL, USA). 36;"

Soluble proteins (10 たg) from cell lysates were separated by 15% SDS-PAGE, and electro-transferred 372"

to PVDF membrane. HA-tagged proteins and DnaK were detected with anti-HA and anti-DnaK 373"

antibodies, respectively. The chemiluminescence signals were developed with the WEST-ZOL® plus 374"

Western Blot Detection System (iNtRON Biotechnology, Gyeonggi-do, South Korea) after the goat 375"

anti-mouse IgG-HRP (Santa Cruz Biotechnology, CA, USA) treatment, and then X-ray film was 376"

exposed to the chemiluminescent light to detect the signals. 377"

378"

Bacterial two-hybrid assay 379"

The protein-protein interaction was determined by the recovery of the adenylate cyclase (CyaA) 37:"

activity through heterodimerization of fusion proteins in Escherichia coli BTH101 reporter strain 37;"


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(cyaA-)"(29). The reporter strain harboring the fusion plasmids pair (e.g., pKT25-rep and pUT18c-ant) 382"

were streaked on LB agar supplemented with Km, Ap and X-gal, or subjected to the く-galactosidase 383"

assay (30) to quantitatively measure the interaction. 384"

385"

Purification of proteins, rTEV protease treatment and analytical size-exclusion chromatography 386"

Cultures of the E. coli BL21 (DE3) harboring pHIS-LexA, -Rep or -Ant (OD600 = ~0.15) were 387"

treated by 100 たM IPTG, and incubated at 25°C for additional 4 hr. Cells were harvested by 388"

centrifugation and lysed in lysis buffer (20 mM Tris pH 8.0, 500 mM NaCl and 20 mM imidazole) by 389"

sonication on ice. Centrifuged (16,000 x g, 4°C for 30 min) and filtered (0.22 たm filter; Millipore, 38:"

Ireland) cell lysate was subjected to the Ni-NTA affinity chromatography (Qiagen, CA, USA) 38;"

according to the manufacturer’s protocol with elution buffer (lysis buffer with 250 mM imidazole). 392"

The eluted protein was concentrated with Vivaspin 20 (3,000 MWCO PES; Sartorius, Goettingen, 393"

Germany) and the buffer was changed (20 mM Tris pH 8.0, 500 mM NaCl and 50% glycerol) using 394"

PD midiTrapTM G-25 (GE healthcare, Buckinghamshire, UK). To remove the His6-tag from the 395"

purified proteins, rTEV protease (1:5 ratios in concentration) was treated for 6 hr at 4°C in a cleavage 396"

buffer (10 mM Tris pH 8.0, 150 mM NaCl, 0.5 mM EDTA and 100 mM DTT). For analytical size-397"

exclusion chromatography, a Superdex 200 10/300 GL column (GE healthcare) was used. The column 398"

was equilibrated with a buffer consisting of 500 mM NaCl and 20 mM Tris pH 8.0, and then purified 399"

proteins (500 たl of 0.8 たg たl-1) were loaded on to the column at a flow rate of 0.5 ml min-1. 39:"

39;"

Electrophoretic mobility shift assay (EMSA) 3:2"

The purified PCR fragments of ant gene promoter region (APR) was け-32P-labeled using T4 3:3"

polynucleotide kinase (Takara, Japan). The labeled DNA (approximately 4 nM) was incubated with 3:4"

varying concentrations of LexA for 30 min at 37°C in 20-たl of reaction mixture containing 1 X 3:5"

binding buffer (10 mM HEPES pH 8.0, 10 mM Tris pH 8.0, 50 mM KCl, 1 mM EDTA, 1 mM 3:6"

dithiothreitol and 5% glycerol) and 1.1 たg of poly(dI-dC). For determination of Rep-binding, various 3:7"

amounts of Rep were incubated for 15 min at 20°C with the 4 nM of labeled DNA in the 20-たl 3:8"


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reaction mixture. When appropriate, Rep was pre-incubated with various concentrations of Ant for 30 3:9"

min at 20°C prior to incubation with labeled DNA. The samples were resolved by 6% native PAGE in 3::"

0.5 X TBE buffer (45 mM Tris-borate pH 8.3 and 1 mM EDTA). The gels were vacuum-dried, and the 3:;"

radioactivity was analyzed using a BAS2500 system (Fuji film, Tokyo, Japan). 3;2"

3;3"

Nucleotide sequence accession number 3;4"

The genome sequences of SPC32H and SPC32N are available at GenBank under accession 3;5"

numbers KC911856 and KC911857, respectively.3;6"


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Results 3;7"

3;8"

Phenotypic and genomic characterization of the two related S. Typhimurium phages SPC32H 3;9"

and SPC32N 3;:"

Previously, we isolated nine phages specific for S. Typhimurium from chicken fecal samples"(15). 3;;"

Two of these phages, which originated from the same sample collection, exhibited distinct plaque 422"

morphologies on a lawn of S. Typhimurium: one phage (SPC32H) formed turbid plaques surrounded 423"

by a halo, but the other phage (SPC32N) formed clear plaques without a halo (Fig. 1A and B). 424"

Transmission electron microscopy (TEM) analysis revealed that both phages belonged to the family 425"

Podoviridae, as they had an isometric head (~62.3 nm in diameter) and a short non-contractile tail 426"

(~15.4 nm in length) with tail shaft and tail spikes (Fig. 1C and D). These two phages infected 427"

identical repertories of Salmonella strains using the O-antigen (O-Ag) of Salmonella as the host 428"

receptor (data not shown). 429"

Sequencing of SPC32H and SPC32N revealed that both phages contain 38,689 base pairs of 42:"

double-stranded DNA with an identical G+C content of 50.16% and 51 predicted open reading frames 42;"

(ORFs) with one Arg-tRNA. About half of the ORFs (24 ORFs) were annotated as hypothetical 432"

proteins, whereas the other annotated proteins were classified into the following modules: DNA 433"

packaging, virion structure morphogenesis, lysogenic conversion, host lysis, and DNA 434"

replication/recombination (Fig. 2A and Table S1). The predicted proteins included a phage integrase 435"

as well as a putative repressor, indicating that both phages might be temperate phages. BlastP searches 436"

revealed that the SPC32H and SPC32N genomes closely resembled Salmonella phage i15 and other 437"

i15-like phages" (31, 32). Indeed, whole genome comparisons made at the DNA level revealed a 438"

significant degree of synteny between the genomes of SPC32H, i15, and the i15-like phage phiV10 439"

(Fig. 2A). In particular, 34 out of 51 SPC32H gene products, including a small/large terminase, a 43:"

head-to-tail joining protein, a putative major coat protein, a putative holin/endolysin, an integrase, a 43;"

repressor, and a putative DNA replication protein, were highly similar (50~100% identity at the amino 442"

acid level) to those of i15 (Table S1). Genes for the putative SPC32H tail structure module (e.g., 443"


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SPC32H_016, 017, and 018) had higher similarity to those of phiV10 than i15 (Fig. 2A). As these 444"

phages infect different hosts (i.e., S. Typhimurium for SPC32H; S. anatum for i15; E. coli O157:H7 445"

for phiV10), differences were observed in the genes encoding tailspike proteins and the flanked 446"

lysogenic conversion module (which converts O-Ag to prevent superinfection). Taken together, these 447"

results suggest that SPC32H and SPC32N should be assigned to the class of i15-like phages. 448"

449"

A single nucleotide change is responsible for the phenotypic difference between the two phages 44:"

Interestingly, a comparison of the full genome sequences of SPC32H and SPC32N revealed only 44;"

two nucleotide differences. One nucleotide difference, designated as m1, is located within the tsp gene 452"

(SPC32H_021), which encodes a phage tailspike, and the other nucleotide difference, m2, is located in 453"

the intergenic region between a gene (SPC32H_020) encoding a hypothetical protein and the tsp gene 454"

(Fig. 2B). To verify whether m1, m2, or both single nucleotide differences were responsible for the 455"

differences between SPC32H and SPC32N, we mutated the SPC32H sequence to match that of 456"

SPC32N. As shown in Fig. 3A, we observed no significant changes in the turbidity of the lysis zone 457"

when the SPC32H m1 sequence was changed to that of SPC32N. However, the turbidity decreased 458"

dramatically when the SPC32H m2 sequence was replaced by that of SPC32N. We therefore 459"

investigated in detail how the single nucleotide difference at the m2 locus leads to this phenotypic 45:"

difference. 45;"

462"

Supplementation with the repressor induces the lysogenic development of the lytic cycle-biased 463"

phage SPC32N 464"

Because lysogen formation is normally associated with plaque morphology, we investigated the 465"

ability of SPC32H and SPC32N to lysogenize. The SPC32H and i15 integrases have 93% identity at 466"

the amino acid level, and both phages contain the highly conserved common core regions and arm-467"

type binding sequences that are required for phage genome integration (31). This suggests that both 468"

phages may integrate their genome into the same attachment site, near the end of the Salmonella guaA 469"

gene. Therefore, to detect lysogenization by SPC32H or SPC32N, we PCR amplified the right end of 46:"


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the phage genome attachment site (attR site) using a primer pair that specifically anneals to the 46;"

upstream region of the phage integrase gene (int) and within the guaA gene. The specific attR band 472"

was amplified from DNA isolated from the SPC32H lysis zone, whereas no band was detected using 473"

DNA isolated from the SPC32N lysis zone (Fig. 3B). The specific attR band was amplified from both 474"

colony and genomic DNA from the putative SPC32H lysogen [〉LT2gtrABC1 (32H)], but not from 475"

DNA isolated from the parental Salmonella strain, SPC32H, or SPC32N (Fig. 3B). Furthermore, the 476"

SPC32H lysogen spontaneously produced phages that formed halo-plaques during prolonged 477"

incubation (data not shown). These results clearly demonstrate that Salmonella can be lysogenized by 478"

SPC32H but not by SPC32N. The specific attR band was amplified from DNA isolated from the lysis 479"

zone of SPC32H m1 but not SPC32H m2 or m12 (Fig. 3C), confirming that m2 is the reason for 47:"

phenotypic differences between SPC32H and SPC32N. 47;"

Because the phage repressor plays a critical role in the maintenance of the lysogenic state by 482"

repressing the expression of lytic genes, and both phages have a putative repressor gene (rep), we 483"

assessed the deficiency of repression in SPC32N. When SPC32H and SPC32N were spotted onto 484"

lawns of a Salmonella strain overexpressing rep (〉LT2gtrABC1+prep), the EOP of both phages was 485"

significantly reduced (<10-5 for SPC32H and <10-2 for SPC32N), and both strains exhibited a more 486"

turbid lysis zone than the control (Fig. 4A). Moreover, the specific attR band was amplified from 487"

DNA isolated from the lysis zone of both phages (data not shown), suggesting that supplementation 488"

with the repressor can promote lysogenic development in SPC32N. These results suggest that 489"

SPC32N is defective in maintaining lysogeny, most likely due to an insufficient amount of the active 48:"

repressor. 48;"

492"

A novel anti-repressor encoded by SPC32H_020 governs the lytic switch 493"

The m2 mutation is located 24-bp upstream of the start codon of the hypothetical protein 494"

SPC32H_020, suggesting that m2 may cause the observed phenotypic differences by affecting the 495"

expression of this protein. SPC32H_020 is a small, 86 amino acid protein with no known conserved 496"

domain or motif. A BlastP search identified 40 hypothetical proteins with more than 64% identity with 497"


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SPC32H_020, but did not identify any protein with a known function. The proteins exhibiting high 498"

homology to SPC32H_020 were from Enterobacteriaceae, such as E. coli, Salmonella spp., 499"

Klebsiella spp., Citrobacter spp., and Cronobacter spp., and from i15-like phages including i15, TL-49:"

2011b, phiV10, SPN1S, and SPN9TCW (Table S2). Interestingly, some larger proteins (>218 amino 49;"

acids) with a relatively low identity (<42%) were annotated as putative anti-repressors, suggesting the 4:2"

possibility of an anti-repressor role for SPC32H_020. 4:3"

To determine whether SPC32H_020 functions as an anti-repressor, we measured the prophage 4:4"

induction efficiency from a Salmonella strain harboring a mutant SPC32H which lacks the gene 4:5"

SPC32H_020 [designated 〉LT2gtrABC1 (32H 〉ant)]. Compared with the WT phage lysogen, the 4:6"

spontaneous induction rate of the mutant phage lysogen was significantly lower (ca. 6 x 10-6-fold 4:7"

lower than WT phage; see Table 4), indicating the critical role of SPC32H_020 in normal prophage 4:8"

induction. Furthermore, MMC treatment did not notably enhance the mutant phage induction (1.11-4:9"

fold increase), but did cause a 78.65-fold increase in induction of the WT phage (Table 4), suggesting 4::"

a potential network between the SPC32H_020 gene and the host SOS response. Because the EOPs in 4:;"

Salmonella of the WT and mutant phage were similar (1.8 x 107 and 4.0 x 107 PFU ml-1, respectively), 4;2"

these results suggest that SPC32H_020 might act as an anti-repressor. The function of the 4;3"

SPC32H_020 gene was further tested by a phage spotting assay using Salmonella harboring a plasmid 4;4"

overexpressing SPC32H_020 from a pBAD promoter (pant). As expected, both SPC32H and SPC32N 4;5"

generated clear lysis zones/plaques (Fig. 4A), and the lysogen-specific attR band was not PCR 4;6"

amplified from DNA isolated from the SPC32H lysis zone (data not shown). To verify the function of 4;7"

SPC32H_020 in lytic switching and prophage induction, an arabinose disc diffusion assay was 4;8"

conducted using Salmonella (〉LT2gtrABC1) and the SPC32H Salmonella lysogen [〉LT2gtrABC1 4;9"

(32H)], both harboring pant. The SPC32H lysogen carrying pant underwent lysis in the presence of 4;:"

15% arabinose, but no lysis was observed in the absence of pant (Fig. 4B). When the arabinose-4;;"

inducible plasmid contained a frame-shifted ant gene (ant*), which was generated by inserting an 522"

additional adenine directly downstream from the SPC32H_020 start codon, the arabinose treatment 523"

did not induce lysis (Fig. 4B), suggesting that lytic-switching is induced by the SPC32H_020 protein 524"


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rather than the RNA. Taken together, our results strongly suggest that SPC32H_020 encodes a novel 525"

anti-repressor protein that plays a significant role in the switch from the lysogenic to the lytic cycle. 526"

We have annotated the SPC32H_020 gene as ant (anti-repressor) and its gene product as Ant. 527"

528"

The m2 sequence in the SOS box causes constitutive expression of the anti-repressor by SPC32N 529"

The results described above indicate that the m2 mutation may allow the overexpression of ant in 52:"

SPC32N. Using the BPROM program, the -10 and -35 sites of the putative ant promoter and one 52;"

LexA-binding site (SOS box), which overlaps the predicted -10 site, were predicted in the upstream 532"

region of the ant gene (Fig. 2B). Intriguingly, m2 is located in the consensus LexA-binding site 533"

sequence (8, 33, 34) (Fig. 2C). LexA is a transcriptional repressor that represses various SOS regulons, 534"

including LexA itself and the RecA protein, via binding to the SOS box. DNA damage induces the 535"

formation of activated RecA nucleoprotein filaments that promote autocleavage of LexA and 536"

consequent derepression of SOS regulons"(8). Therefore, we hypothesized that the ant gene is an SOS 537"

regulon controlled by LexA and that m2 in the consensus LexA-binding site sequence might prevent 538"

the LexA-mediated repression of the ant gene in SPC32N. 539"

To test this hypothesis, we first examined the promoter activity of the ant gene in both SPC32H 53:"

and SPC32N via a bioluminescence reporter assay using luxCDABE. In contrast to the low RLU 53;"

(relative light units) detected using the ant promoter from SPC32H (Pant_H), the promoter from 542"

SPC32N (Pant_N) exhibited approximately 2-log higher values (Fig. 5A and B). Treatment with MMC 543"

significantly increased the RLUs produced from a clone harboring pPant_H::lux, but not in a clone 544"

harboring pPant_N::lux (Fig. 5A and B), suggesting that Pant_H expression was activated by DNA 545"

damage, whereas Pant_N was expressed constitutively, and independent of DNA damage. To elucidate 546"

whether these responses were associated with LexA, we constructed Salmonella mutants without the 547"

lexA gene or expressing a non-cleavable form of LexA [lexA(G85D)] and measured the 548"

bioluminescence from the reporter plasmid pPant_H::lux. Both mutants were constructed in a 〉sulA 549"

background to suppress the lethality of the lexA deletion (35). This sulA deletion did not affect the 54:"

reporter gene expression (data not shown). In the absence of lexA, the Pant_H activity was comparable 54;"


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to that observed in the lexA+ background in the presence of MMC, and the Pant_H activity was not 552"

affected by MMC treatment (Fig. 5B lexA-). In addition, the lexA+ phenotype was partially rescued by 553"

in trans complementation of lexA (data not shown). In contrast, replacing LexA with the non-554"

cleavable form of LexA prevented promoter activation by MMC (Fig. 5B lexA(G85D)), indicating 555"

that DNA damage activates the ant promoter through LexA proteolysis. The results were similar 556"

regardless of the presence of SPC32H prophage (Fig. 5B lexA+ vs lexA+(32H)), suggesting that no 557"

other factors, including the SPC32H repressor, are involved in ant gene regulation, despite the 558"

presence of a repressor-binding site immediately upstream of ant promoter (Fig. 2B and see below). 559"

We next performed an electrophoretic mobility shift assay (EMSA) to show the binding of LexA 55:"

to the SOS box within Pant_H or Pant_N. When the radio-labeled DNA fragment APRH* (ant gene 55;"

promoter region from SPC32H) was incubated with an increasing amount of purified Salmonella 562"

LexA, a specific mobility shift was observed, and the APRH* fragment was released by the addition of 563"

the unlabeled competing cold probe APRH (Fig. 5C lanes 1 to 8). In contrast, the unlabeled cold probe 564"

APRN (ant gene promoter region from SPC32N) was unable to compete with APRH* for LexA, and 565"

the APRN* fragment was not shifted in the presence of LexA (Fig. 5C lanes 9 to14), confirming that 566"

LexA cannot repress ant expression in SPC32N due to an inability to bind to the SOS box containing 567"

the m2. 568"

Based on these results, we investigated the overall cascade of SPC32H induction using 569"

Salmonella strains lysogenized by a derivative of SPC32H containing lacZ transcriptionally fused to 56:"

the putative recET genes. Because the phage recE and recT gene products, a 5’å3’ exonuclease and a 56;"

single-strand DNA binding/annealing protein, respectively, promote homologous recombination to 572"

mediate the integration/excision of phage genome to/from the host chromosome" (36-38), the 573"

expression of recET (and its orthologous genes) can be used as a reporter for prophage induction. As 574"

shown in Fig. 6, treatment with MMC, but not other antibiotics, activated the recET::lacZ fusion in 575"

the lexA+ background but not in the lexA(G85D) background, indicating that the DNA damage 576"

generated by MMC induces SPC32H induction dependent on LexA proteolysis. The expression of Ant 577"

clearly resulted in lysogen-specific lysis (Fig. 4B), supporting the idea that derepression of ant gene 578"


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via MMC-induced LexA proteolysis leads to phage induction. Taken together, these results 579"

demonstrate that the ant gene of SPC32H is negatively regulated by LexA and that the m2 in the SOS 57:"

box causes the dramatic phenotype differences between SPC32H and SPC32N by influencing ant 57;"

expression. 582"

583"

The anti-repressor Ant interacts directly with the cognate repressor Rep 584"

The putative repressor from SPC32H (designated Rep), encoded by SPC32H_041, is a 198-585"

amino-acid protein that contains a helix-turn-helix motif. The RecA-mediated autocleavage site (Ala-586"

Gly or Cys-Gly), a highly conserved site in cleavable repressors such as lambda CI"(39), is not present 587"

in SPC32H Rep, strongly supporting the notion that SPC32H prophage induction involves the 588"

inhibition of Rep through means other than autocleavage assisted by RecA nucleofilaments. The 589"

immunodetection of HA epitope-tagged Rep demonstrated that the expression level of Rep was 58:"

remained virtually constant (i.e., was not cleaved) throughout a one-hour treatment with MMC (Fig. 58;"

7A). The lytic switch was activated by MMC in this experiment, as shown by the fact that HA-tagged 592"

Ant was expressed and accumulated after treatment with MMC (Fig. 7A lower panel). The HA-tagged 593"

versions of the Rep and Ant proteins are fully functional (data not shown). 594"

To explore the possible interaction between Rep and Ant, we performed a bacterial two-hybrid 595"

assay based on the restoration of く-galactosidase activity in E. coli cyaA mutant strain BTH101 (29). 596"

The reporter strain E. coli BTH101 expressing the combination of hybrid proteins (i.e., T25-Rep/T18-597"

Ant or T25-Ant/T18-Rep) produced blue colonies on X-gal plates (data not shown) and exhibited a 598"

significantly higher (approximately 10 to 50-fold) level of く-galactosidase activity than the negative 599"

control (i.e., E. coli BTH101 expressing the unfused T18 and T25 peptides) (Fig. 7B), indicating a 59:"

heterodimerization of the hybrid proteins via interaction between Rep and Ant. Strong Rep::Rep and 59;"

Ant::Ant interactions were also observed, implying the possibility of multimerization by each protein. 5:2"

Indeed, the results of analytical size-exclusion chromatography demonstrated that Rep and Ant were 5:3"

able to dimerize and tetramerize, respectively (data not shown). Taken together, these results suggest 5:4"

that Rep and Ant can interact with themselves and each other. 5:5"


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EMSA using purified Rep and Ant revealed that Ant inhibits Rep target site binding. The high 5:6"

homology (97% identity) between SPC32H Rep and the i15 repressor suggests that Rep may 5:7"

recognize the same DNA sequence (5’-" ATTACCnnnnGGTAAT -3’) as the i15 repressor. 5:8"

Radiolabelled APRH*, which includes the putative repressor-binding site as well as the SOS box, was 5:9"

also used in this assay. Two DNA-protein complex bands with different mobilities appeared when the 5::"

purified Rep was incubated with APRH* (Fig. 7C lanes 1 to 4), suggesting that APR may have two 5:;"

Rep-binding sites with different affinities for Rep. A competition assay using a non-labeled cold probe 5;2"

demonstrated the specificity of Rep-binding for APR (Fig. 7C lanes 5 and 6). Notably, pre-incubation 5;3"

of Rep with purified Ant prevents the mobility shift of the APRH* fragment in an Ant concentration-5;4"

dependent manner (Fig. 7C lanes 7 to 9), suggesting that the specific interaction between Ant and its 5;5"

cognate repressor Rep interferes with Rep binding to its target DNA. Note that the protein 5;6"

concentrations indicated were calculated based on the assumption that the Rep and Ant stocks 5;7"

consisted entirely of active dimers and tetramers, respectively. The APRH* fragment clearly did not 5;8"

exhibit a mobility shift when incubated with Ant alone (Fig. 7C lane 10), excluding the possibility that 5;9"

Ant inhibits Rep activity by competing for the Rep-binding site with Rep. 5;:"

5;;"


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3:""

Discussion 622"

The goal of this study was to determine the cause for the phenotypic differences between two 623"

highly similar podoviral i15-like phages, SPC32H and SPC32N. We detected two nucleotide 624"

differences between the two phage genomes, but only one, located within a non-coding region, was 625"

responsible for the phenotypic differences. This nucleotide polymorphism, m2, was located within a 626"

consensus LexA-binding site sequence that overlaps the -10 site of the promoter for SPC32H_020, 627"

which encodes a hypothetical protein (Fig. 2). This sequence difference prevents LexA from binding 628"

to its binding site, allowing the constitutive expression of a small hypothetical protein (Fig. 5), which 629"

we have identified as a novel anti-repressor of the family Podoviridae. This anti-repressor inhibits the 62:"

binding of its cognate phage repressor to regulatory regions (Fig. 7C), resulting in a switch of the 62;"

phage lifecycle from lysogenic to lytic. 632"

To date, at least two categories of lytic switch anti-repression systems have been identified in 633"

temperate phages. The first system is represented by the Cro protein of several lambdoid phages, such 634"

as phage lambda, HK022, and HK97. In this system, the binding of the Cro protein to target operator 635"

sites prevents expression of the cI gene that encodes the phage repressor CI" (40). In contrast, the 636"

second system controls the repressor activity at the protein level. For example, the anti-repressor Tum 637"

from myoviral coliphage 186 binds directly to the phage repressor CI, preventing CI from binding to 638"

its operator sites (12). Notably, the latter system has been reported in only a few temperate phages, 639"

including siphoviral coliphage N15 (11) and the siphoviral prophages Gifsy-1and Gifsy-3 identified in 63:"

S. Typhimurium strain 14028 (10). In the present study, we have elucidated the mechanism and 63;"

regulation of an anti-repressor, which belongs to the second category of lytic switch anti-repression 642"

systems, and is the first example of this type of system in the Podoviridae family of the order 643"

Caudovirales. A notable common feature of this second system type is the LexA-regulated initiation 644"

of anti-repressor expression. Although the phage P22 also produces an anti-repressor that inactivates 645"

the c2-repressor and prevents RecA-dependent c2 proteolysis (41, 42), it is unknown whether LexA 646"

regulates the expression of the anti-repressor. However, the presence of a consensus LexA-binding 647"

sequence 38-bp upstream of the start codon of the anti-repressor protein suggests that LexA may be 648"


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involved in the regulation of the P22 anti-repressor and, consequently, that the P22 anti-repressor may 649"

be a member of the second category of anti-repression systems. 64:"

Linking the host SOS response to the lytic switch is a fundamental strategy used by prophages to 64;"

escape from damaged host cells. Compared to the RecA-dependent cleavable repressor system such as 652"

the lambda CI (7, 40), the anti-repression system appears to be more advantageous to the prophages. 653"

If host bacteria are able to repair DNA damage before prophage induction and survive (43), it would 654"

be more beneficial for the prophages to remain in the host cell. If lysis occurred, the induced phages 655"

would need to re-establish the prophage state in new host bacterial cells to stably maintain their 656"

genome as a part of a host genome. This superfluous step could easily be prevented by expressing the 657"

anti-repressor in a LexA cleavage-dependent manner. As anti-repressor levels are reduced by the 658"

replenished LexA pool, lysogenic development could resume because inactivated, rather than 659"

degraded, repressors can be restored to function by dissociating from the anti-repressor. Although we 65:"

did not demonstrate the reversible binding of Ant and the recovery of Rep activity after Ant 65;"

dissociation in the present study, Rep levels were stably maintained without degradation during MMC 662"

treatment (Fig. 6), suggesting that recycled Rep could be used in the resumed SPC32H lysogenic 663"

development. Indeed, the anti-repressor Tum/repressor CI pair from coliphage 186 exhibits reversible 664"

Tum-binding and the recovery of CI activity after dissociation from Tum" (12). We are currently 665"

attempting to elucidate this issue by investigating the structure of the Rep-Ant complex as well as the 666"

individual proteins. 667"

Considering the advantages of rapid resumption of the regulatory circuit, it is possible that this 668"

type of repressor/anti-repressor system is widespread among the temperate phages. Remarkably, 669"

several homologues of SPC32H Ant (38-100% amino acid similarities) were identified in other 66:"

Podoviridae phages and various bacteria in the family Enterobacteriaceae (Table S2), most likely as a 66;"

gene product of unknown function of prophages. Moreover, the phage anti-repressors Tum (from 672"

Myoviridae coliphage 186), AntC (from Siphoviridae coliphage N15), GfoA (from Siphoviridae phage 673"

Gifsy-1) and Ant (from Podoviridae phage SPC32H) are distinct from each other at the amino acid 674"

sequence level (Fig. 8), suggesting that diverse repressor/anti-repressor pairs are present in the order 675"


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Caudovirales to allow for more prudent control of lytic/lysogenic switching. Therefore, as suggested 676"

by Mardanov and Ravin, the cleavable repressor system may not be the exclusive mechanism for 677"

lytic/lysogenic regulation in temperate phages (11). As recently illustrated by Lemire et al., anti-678"

repressors can mediate crosstalk between prophages in polylysogenic strains (10). Thus, further 679"

studies regarding the trans-activity of diverse phage anti-repressors, including SPC32H Ant, would 67:"

provide insight into the coordinated behavior of temperate phage subversion of their bacterial prey. 67;"

682"

683"

684"

685"

Acknowledgments 686"

This work was supported by a National Research Foundation of Korea (NRF) grant funded by 687"

the Ministry of Education, Science and Technology (No. 20090078983). 688"

689"


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43""

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high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 79;"

177:4121-4130. 7:2"

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7:6"

7:7"

7:8"


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Tables 7:9"

Table 1. Bacterial strains and bacteriophages used in this study 7::"

Strains Relevant characteristicsa Reference or source

Bacterial strains

Salmonella enterica serovar Typhimurium

LT2(c)

Prophage cured strain LT2; wild-type; host for phage

SPC32H and SPC32N

(13)

SR5003 LT2(c) with 〉LT2gtrABC1 (14)

SR5100

〉LT2gtrABC1 (32H); SR5003 lysogenized by

SPC32H

This study

SR5158 LT2(c) with 〉LT2gtrABC1 〉sulA 〉lexA This study

SR5176

LT2(c) with 〉LT2gtrABC1 〉sulA lexA(G85D); non-

cleavable LexA mutant

This study

SR5189

〉LT2gtrABC1 (32H recET::lacZ); SR5003

lysogenized by SPC32H recET::lacZ

This study

SR5188

〉LT2gtrABC1 〉sulA lexA(G85D) (32H recET::lacZ);

SR5176 lysogenized by SPC32H recET::lacZ

This study

SR5192

〉LT2gtrABC1(32H rep-HA); SR5003 lysogenized by

SPC32H rep-HA

This study

SR5197

〉LT2gtrABC1(32H rep-HA ant-HA); SR5003

lysogenized by SPC32H rep-HA ant-HA

This study

Escherichia coli

BTH101

F- cya-99 araD139 galE15 galK16 rpsL1(Strr) hsdR2

mcrA1 mcrB1; reporter strain in bacterial two-hybrid

assay

(29)

BL21(DE3)

F- ompT hsdS (rB- mB

-) gal (DE3); protein

overexpression

Laboratory collection


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Bacteriophages

SPC32H Infect S. Typhimurium; O-antigen-specific This study

SPC32N Infect S. Typhimurium; O-antigen-specific This study

SPC32H 〉ant SPC32H derivative with 〉ant This study

SPC32H m1

SPC32H derivative with one nucleotide substitution

in tsp gene (C to T)

This study

SPC32H m2

SPC32H derivative with one nucleotide substitution

in ant gene promoter (G to T)

This study

SPC32H m12

SPC32H derivative with two nucleotide substitutions

in tsp gene (C to T) and ant gene promoter (G to T)

This study

a Strr, streptomycin resistant. 7:;"

7;2"


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4:""

Table 2. Plasmids used in this study 7;3"

Plasmids Relevant characteristicsa Reference or source

Gene overexpression

pBAD24 General expression vector with the PBAD promoter; Apr (44)

prep pBAD24-rep; Apr This study

pant pBAD24-ant; Apr This study

Pant* pBAD24-ant*; encoding frame-shifted ant;Apr This study

Luciferase reporter assay

pBBRlux

A derivative of broad-host-range cloning vector

pBBR1MCS containing a promoterless luxCDABE; Cmr

(28)

pPant_H::lux pBBRlux with ant promoter of SPC32H; Cmr This study

pPant_N::lux pBBRlux with ant promoter of SPC32N; Cmr This study

Bacterial two-hybrid assay

pKT25 Encodes T25 fragment of adenylate cyclase; Kmr (29)

pUT18C Encodes T18 fragment of adenylate cyclase; Apr (29)

pKT25-zip pKT25 with zip; Kmr (29)

pUT18C-zip pUT18C with zip; Apr (29)

pKT25-rep pKT25 with rep; Kmr This study

pUT18C-ant pUT18C with ant; Apr This study

pKT25-ant pKT25 with ant; Kmr This study

pUT18C-rep pUT18C with rep; Apr This study

Protein expression

pHIS-parallel1

Protein expression vector; allowing N-terminal His6-tagging

with a TEV cleavage site; Apr

(45)

pHIS-LexA pHIS-parallel1 with lexA; Apr This study

pHIS-Rep pHIS-parallel1 with rep; Apr This study

pHIS-Ant pHIS-parallel1 with ant; Apr This study

a Kmr, kanamycin resistant; Apr, ampicillin resistant; Cmr, chloramphenicol resistant 7;4"

7;5"


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Table 3. Oligonucleotides used in this study 7;6"

Oligonucleotides Sequences (5’s3’)a Purpose

32H-attP ATA CAG TTT GTC CTG CGG CTT GAG PCR for attR site

32H-attB GAC AAA GTC AGT CAG GCG TTT ACC PCR for attR site

32H-rep-CF2 AAT GGG CAA ATG AAT TCG CTA TGA AA prep construction

32H-rep-CR1 TGG TAA TTG CGT GTC GAC TGA G prep construction

32H-ant-CF1 TGT TTG CAT GGA GAA TTC GAG ATG C pant construction

32H-ant-CR1 GCC GCA GAG TCG ACC TTT TAT TTT T Pant, Pant* construction

32H-ant*-CF2 CAT GGA GAA TTC GAG ATG ACA ACG G Pant* construction

32s-Pant-CF1 TCA GTT GAG CTC GTC ATG TAA G pPant_H::lux and pPant_N::lux

construction

32s-Pant-CR1 GGT GAT ACT GCC ACT AGT TCT C pPant_H::lux and pPant_N::lux

construction

32H-recE-lacZ-Red-F AGT AAG TCA CAA CTG GAT ATG GTG GCC

AAG AAC CCT TCC CTG TAG GCT GGA GCT

GCT TCG

SR5188 and SR5189

construction

32H-recT-lacZ-Red-R TCT TTG TTG CAG GTG TGG CGC CGT GGC

GCC ACG GTG GTG A AT TCC GGG GAT CCG

TCG ACC

SR5188 and SR5189

construction

32H-rep-HA-Red-F CTC ATT CAT AAA CTT CGT GTT TGA GCA GAA

CAA AAG CAA GTA TCC GTA TGA TGT TCC TGA

TTA TGC TAG CCT CTA ATG TAG GCT GGA GCT

GCT TCG

SR5192 and SR5197

construction

32H-rep-HA-Red-R ACC GCC ATC GGG CAG GTA AAG CGT CAG

AAT GGC AGG GGA TAT TCC GGG GAT CCG TCG

ACC

SR5192 and SR5197

construction

32H-ant-HA-Red-F TCT CGA TTA CTG TGC AGA ACA GTT ACG AAA

ACA AAC CAC ATA TCC GTA TGA TGT TCC TGA

TTA TGC TAG CCT CTA ATG TAG GCT GGA GCT

SR5197 construction


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GCT TCG

32H-ant-HA-Red-R TGT CAT AGC ATG AAT GTG ACA TGT CAC GAG

GCC GCA GAA GAT TCC GGG GAT CCG TCG

ACC

SR5197 construction

pKT25-32H-rep-CF1 AAC TGC AGG GAT GAA AAG TAT TTA TGA CAT pKT25-rep construction

pKT25-32H-rep-CR1 CGG GAT CCT TAC TTG CTT TTG TTC TG pKT25-rep and pUT18C-

rep construction

pUT18C-32H-ant-CF1 AAC TGC AGG ATG CAA CGG CAG TAT CA pUT18C-ant construction

pUT18C-32H-ant-CR1 CGG GAT CCT TAT GTG GTT TGT TTT CGT AAC pUT18C-ant and pKT25-

ant construction

pKT25-32H-ant-CF1 AAC TGC AGG GAT GCA ACG GCA GTA TCA pKT25-ant construction

pUT18C-32H-rep-CF1 AAC TGC AGG ATG AAA AGT ATT TAT GAC ATA

AG

pUT18C-rep construction

pHIS-LT2-lexA-CF2 TAT ATA CAC CCC ATG GGC GGA ATG AAA G pHIS-LexA construction

pHIS-LT2-lexA-CR2 ATT GCC GGA TCT CGA GTT ACA AGG AG pHIS-LexA construction

pHIS-32H-Rep-CF1 AAT ACC ATG GCT ATG AAA AGT ATT TAT GAC

ATA AGA CGC

pHIS-Rep construction

pHIS-32H-Rep-CR1 AAA GCT CGA GAA TGG CAG GGG ATT ACT

TGC

pHIS-Rep construction

pHIS-32H-Ant-CF4 CAT AAG CCA TGG GGA TGC AAC GGC AGT

ATC AC

pHIS-Ant construction

pHIS-32H-Ant-CR3 GCC GCA GAC TCG AGC TTT TAT TTT TCA TTA

TGT GG

pHIS-Ant construction

32s-APR-CF1 CTT CAG TTG AGA CCG TCA TG PCR for APRH and APRN

32s-APR-CR1 TAA GAT GTG AGT CCT CCA CC PCR for APRH and APRN

a Restriction enzyme sites and HA-tag coding sequences are underlined and italicized, respectively. 7;7"

Bold case indicates the artificially inserted redundant nucleotide to generate the frame-shift in ant 7;8"

gene.7;9"


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Table 4. Comparison of prophage induction efficiency 7;:"

Strain

Phage titer (PFU ml-1

) Fold change

(fold) - MMC + MMC (1 たg ml-1)

〉LT2gtrABC1 (32H) 4.45 x 108 3.50 x 1010 78.65

〉LT2gtrABC1 (32H 〉ant) 2.65 x 103 2.95 x 103 1.11

7;;"


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Figure legends 822"

823"

Fig. 1. Two similar S. Typhimurium-specific Podoviridae phages, SPC32H and SPC32N, produce 824"

morphologically distinct plaques. 825"

A and B. Plaque morphology of SPC32H (A) and SPC32N (B). Dilutions (10 たl) of each phage stock 826"

were spotted onto a lawn of S. Typhimurium LT2 (c) 〉LT2gtrABC1 strain (SR5003). 827"

C and D. TEM images of SPC32H (C) and SPC32N (D). Insets at the bottom left of each panel show 828"

the enlarged virion morphology with a black scale bar (50 nm). White arrow and arrow heads indicate 829"

the tail shaft and tail spikes, respectively. 82:"

82;"

Fig. 2. There are two single nucleotide differences between the genomes of i15-like phage SPC32H 832"

and SPC32N. 833"

A. DNA alignment of the genomes of phage i15 (NC_004775.1), SPC32H, and phiV10 834"

(NC_007804.2) using Easyfig. High sequence similarity between the genomes is indicated by the gray 835"

regions. SPC32H ORFs are indicated by numbered or annotated arrows. Phage functional modules are 836"

indicated under the arrows. ant, anti-repressor; tsp, tailspike; oac, O-acetyltransferase; hol, holin; end, 837"

endolysin; int, integrase; rep, repressor. Note that the SPC32N genome is identical to that of SPC32H 838"

with the exception of two single nucleotide differences (see panel B). 839"

B. Schematic representation of the location of the two single nucleotide differences, m1 and m2. The 83:"

partial SPC32H genome sequence surrounding the two single nucleotide differences is shown. m1 83;"

(located within the tsp gene) and m2 (located in the intergenic region between SPC32H_020 and tsp) 842"

are indicated in bold, upper-case letters. The predicted -10 and -35 sites of the putative promoter for 843"

SPC32H_020 gene are boxed. The putative LexA-binding site (SOS box) and the putative repressor-844"

binding site are underlined and double-underlined, respectively. 845"

C. Consensus sequence of the LexA-binding site from E. coli (8, 33, 34) and the putative LexA-846"

binding sites from phage SPC32H and SPC32N. m2 is indicated with a gray background. Note that 847"

the LexA-binding site sequences for SPC32H and SPC32N shown here are reverse complements of 848"


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the sequence shown in panel A. 849"

84:"

Fig. 3. Introduction the m2 sequence from SPC32N induces SPC32H to enter the lytic cycle. 84;"

A. High titer phage stock (>107 PFU ml-1; 10 µl) of SPC32H, SPC32N and three mutant phages 852"

derived from SPC32H were spotted onto a lawn of S. Typhimurium LT2 (c) 〉LT2gtrABC1. 853"

B. SPC32H can lysogenize host Salmonella, whereas SPC32N cannot. Various template samples 854"

were PCR amplified with an attR-specific primer pair. M, DNA marker 1 Kb+ (Invitrogen); i, inner 855"

part of the lysis zone; e, edge of the lysis zone; gDNA, genomic DNA; 〉LT2gtrABC1(32H), SPC32H 856"

lysogen (SR5100). 857"

C. DNA isolated from the lysis zones shown in panel A was PCR amplified using primers specific 858"

for the attR site to determine the lysogenization of each phage. Lanes 1 to 5 correspond to each lysis 859"

zone shown in panel A. Note that the introduction of m2 resulted in a disappearance of the lysogen-85:"

specific attR band (lanes 4 and 5). 85;"

862"

Fig. 4. The novel anti-repressor, encoded by SPC32H_020 (ant), induces the lytic development of 863"

SPC32H. 864"

A. Supplementation with the putative repressor leads to the lysogenic development of the lytic cycle-865"

biased phage SPC32N, while supplementation with the putative anti-repressor results in the lytic 866"

development of SPC32H. Salmonella strains transformed with a control plasmid (pBAD24), a 867"

putative repressor-overexpressing plasmid (prep) or an SPC32H_020-overexpressing plasmid (pant) 868"

were infected serially diluted (10-fold) stocks of SPC32H or SPC32N. L-arabinose (0.2%, final 869"

concentration) was added to induce SPC32H_020 expression from pant. 86:"

B. The expression of the SPC32H_020 protein promotes the switch from lysogenic to lytic 86;"

development. The SPC32H lysogen [〉LT2gtrABC1 (32H); SR5100] and non-lysogen (〉LT2gtrABC1; 872"

SR5003) strains were transformed with pant or a control plasmid (pBAD24), and the resulting strains 873"

were subjected to a disc diffusion assay with 10 µl of 15% L-arabinose. pant* indicates the plasmid 874"

encoding a frame-shifted ant gene. Arabinose-induced bacterial lysis was observed only in the 875"


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SPC32H lysogen harboring pant. 876"

877"

Fig. 5. The ant promoter of SPC32H is activated by DNA damage via LexA proteolysis, whereas the 878"

SPC32N ant promoter is constitutively active, due to the inability of LexA to bind to the m2-879"

containing consensus LexA-binding site. The RLU (relative light units) were calculated by dividing 87:"

the measured bioluminescence by the A600 value. The mean and SD of three independent assays are 87;"

shown on a log-scale on the Y-axis (A and B). 882"

A. Time-course observation of the ant promoter activity in the presence or absence of DNA damage. 883"

Salmonella strains harboring the bioluminescence reporter plasmid pPant_H::lux (luxCDABE fused to 884"

the putative ant promoter of SPC32H) or pPant_N::lux (luxCDABE fused to the putative ant promoter of 885"

SPC32N) were incubated at 37°C, and the bioluminescence, as well as the A600 of the culture, was 886"

measured every half hour. The vertical arrows indicate MMC treatment (1 たg ml-1, final 887"

concentration; 3 hr after incubation). 888"

B. The ant promoter activity of the various Salmonella strains at A600 = ~0.6 harboring the 889"

bioluminescence plasmid. MMC (1 たg ml-1, final concentration) was added after 3 hr of incubation. 88:"

lexA+, 〉LT2gtrABC1, SR5003; lexA+(32H), 〉LT2gtrABC1(32H), SR5100; lexA-, 〉LT2gtrABC1 〉sulA 88;"

〉lexA, SR5158; lexA(G85D), 〉LT2gtrABC1 〉sulA lexA(G85D), SR5176. ***, P < 0.001. 892"

C. LexA specifically binds to the putative ant gene promoter region of SPC32H but not to that of 893"

SPC32N, which contains m2. The け-32P-labeled DNA fragment of the ant gene promoter region from 894"

SPC32H (APRH*) or from SPC32N (APRN*) was incubated with the indicated amounts of purified 895"

Salmonella LexA and was subjected to an electrophoretic mobility shift assay (EMSA). 896"

Corresponding unlabeled DNA fragments (APRH and APRN) were used for the competition analysis. 897"

The position of the unbound fragments (F) and fragments retarded by LexA binding (B) are indicated. 898"

899"

Fig. 6. DNA damage-induced LexA proteolysis followed by SPC32H ant expression induces the 89:"

switch to lytic development. The lacZ gene, transcriptionally fused to the putative recET genes, was 89;"

introduced into the SPC32H lysogens harboring an intact (lexA+) or non-cleavable (lexA(G85D)) 8:2"


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LexA, and the resulting strains were subjected to a disc diffusion assay with the following solutions: 8:3"

MMC, 0.5 mg ml-1 mitomycin C; Cm, 2.5 mg ml-1 chloramphenicol; Ap, 10 mg ml-1 ampicillin; and 8:4"

D.W., distilled water. Note that the blue zone appears to surround the MMC disc in the lexA+ 8:5"

background only. 8:6"

8:7"

Fig. 7. DNA damage induces Ant accumulation, but not Rep cleavage, and the consequent binding of 8:8"

Ant to Rep inhibits the binding of Rep to specific operators. 8:9"

A. Salmonella strains lysogenized by SPC32H expressing HA-tagged Rep (upper panel; 8::"

〉LT2gtrABC1 (32H rep-HA), SR5192) or both HA-tagged Rep and HA-tagged Ant (lower panel; 8:;"

〉LT2gtrABC1 (32H rep-HA ant-HA), SR5197) were exposed to MMC for 1 or 2 hr, respectively. The 8;2"

MMC-treated bacterial cultures were sampled at the indicated time points and subjected to the 8;3"

Western blotting to immunodetect the HA-tagged proteins. DnaK was used as an internal control. 8;4"

B. Bacterial two-hybrid assays revealed the direct binding of Ant to Rep. The く-galactosidase 8;5"

activity of E. coli BTH101 reporter strains harboring the indicated plasmid pairs were measured. The 8;6"

activities are presented in Miller units. B, a backbone plasmid. 8;7"

C. EMSA with purified Rep and Ant demonstrates the Ant-mediated inhibition of Rep-binding to its 8;8"

operators. Mixtures of APRH* and the indicated amounts of Rep were incubated at 20°C for 15 min in 8;9"

1 X binding buffer supplemented with 1.1 たg of poly(dI-dC) and then electrophoresed on a 6% native 8;:"

acrylamide slab gel for EMSA. For competition analysis, unlabeled APRH fragments were added as 8;;"

cold probes to the mixture. When appropriate, Rep was pre-incubated with the indicated amounts of 922"

Ant at 20°C for 30 min, and further incubated with APRH* as described above. The positions of the 923"

unbound fragments (F) and fragments retarded by Rep binding (B1 and B2) are indicated. 924"

925"

Fig. 8. Amino acid alignment of the phage anti-repressors. The amino acid sequences of Tum (from 926"

coliphage 186), AntC (from coliphage N15), GfoA (from Gifsy-1) and Ant (from SPC32H) were 927"

aligned using ClustalW2. There are no noticeable consensus residues, demonstrating the diversity of 928"

phage anti-repressors in the order Caudovirales. 929"


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anti-repression system associated with the life cycle...

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