Determination of the attP and attB sites of phage wCD27 from Clostridium difficile NCTC 12727

The attP region of the Clostridium difficile phage wCD27 was identified, located immediately downstream of the putative recombinase. The phage could integrate into two specific sites (attB) in the C. difficile genome, one of which was in an open reading frame encoding a putative ATPase of an ABC transporter and the other in an open reading frame encoding a putative ATPase of the flagella protein export apparatus. The prophage was capable of excision and formation of a circular molecule and phages were spontaneously released at a low frequency during growth. Infection and lysogeny of a C. difficile strain previously shown to be sensitive to wCD27 were demonstrated, leading to a reduction in toxin production. Finally, a putative repressor was identified which is likely to be involved in maintaining lysogeny in these strains.


INTRODUCTION
Clostridium difficile infection is the most common cause of antibiotic-associated colitis and represents a considerable burden on healthcare services worldwide (Lo Vecchio & Zacur, 2012).Sequenced C. difficile strains contain an array of mobile genetic elements including plasmids, conjugative transposons and temperate bacteriophages (phages) (Sebaihia et al., 2006;Stabler et al., 2009;Brouwer et al., 2011).The integration of phage into host DNA during lysogeny can be beneficial to both the phage and the host bacterium.By sparing the host cell, the phage can replicate at the rate of the bacterial replicon before releasing progeny that can go on to infect further cells.Genetic elements encoding virulence factors such as toxin genes and antibiotic resistance are often embedded in phage DNA (Bishai & Murphy, 1988;Chen et al., 2011) and acquisition can impart significant competitive advantages, enabling the host bacterium to survive in changing environments.In some Clostridium spp.toxins are also encoded by phage sequences (Eklund et al., 1971) and some homology has been reported between proteins involved in C. difficile toxin production and proteins encoded by C. difficile phages (Goh et al., 2005;Tan et al., 2001).
Phage wCD27 has been completely sequenced (Mayer et al., 2008) and shown to belong to the family Myoviridae, the genome size being 50 930 bp.The phage has several modules which are related to other C. difficile phages.In the current work, we identify the wCD27 attP region and show that the phage can integrate into two specific sites (attB) in the C. difficile genome.Furthermore, we show that in the prophage state a candidate repressor is expressed which may be involved in the maintenance of lysogeny and modulation of toxin production.
Production of a lysogenic C. difficile strain.wCD27 was induced from a fresh turbid culture of NCTC 12727 with 3 mg mitomycin C ml 21 .Phage particles were harvested by centrifugation for 12 min at 1500 g and the supernatant filtered through a 0.45 mm membrane.500 ml of this suspension containing greater than 10 7 p.f.u ml 21 was spotted onto a lawn of NCTC 11209 to give what appeared to be confluent lysis.The plate was incubated for 24-36 h and bacteria from the centre of the zone of lysis were subcultured.After propagation, the presence of prophage was confirmed by mitomycin C induction and a subsequent plaque assay.
Measurement of toxin production.Liquid cultures of NCTC 11209, and its lysogen containing wCD27, were grown in brain heart infusion (BHI) broth with supplements until they reached an optical density (OD 600 ) of 1.5.Supernatants were harvested by centrifugation at 1500 g for 12 min and tested using the C. difficile Premier Toxins A IP: 54.70.40.11 On: Sat, 03 Aug 2019 09:11:07 & B ELISA immunoassay (Meridian Bioscience) according to the manufacturer's instructions.Plate washing was performed manually and the optical density of samples was measured at 450 nm using a plate reader (Thermomax, Molecular Devices).The toxin assay results of the wild-type strains relative to the lysogen were compared and statistically analysed using Student's t-test.
Determination of the site of wCD27 integration and detection of circularized phage genome.To determine the insertion site of wCD27 in C. difficile, single-specific-primer PCR (SSP PCR) with primers ORF41-F and ORF42-R was used.The binding sites of the primers are shown in Fig. 1.Genomic DNA was extracted from C. difficile NCTC 12727 using Qiagen Genomic Tips (Qiagen), with the addition of 50 U mutanolysin ml 21 (Sigma) to aid cell lysis.Genomic DNA and pUC19 were digested with HindIII and ligated overnight with T4 DNA ligase (New England Biolabs) at 16uC.2.5 ml of the ligation mix was used as a template in a PCR using wCD27-specific primers (ORF41-F or ORF42-R) and pUC19-specific primers (pCC1F or pCC1R) (Table 1).Sequenced PCR products were analysed using NCBI tools (http://www.ncbi.nlm.nih.gov/).The integration sites were confirmed by PCR using the primer pairs detailed in Table 1 and NCTC 12727 genomic DNA as template.
To detect phage and regenerated target sites from individual colonies, cells were resuspended in 30 ml dH 2 O and 5 ml was used as a template in 25 ml PCRs using MyTaq Red Mix (Bioline) and the primer pairs detailed in Table 1.
Determination of uninduced release of phage particles.200 ml aliquots of a fresh overnight culture of C. difficile NCTC 12727 were used to inoculate eight pre-reduced 25 ml BHI broths containing supplements.At hourly intervals, single cultures were harvested and used to enumerate the level of C. difficile from serial dilutions in prereduced PBS, which were plated in triplicate onto BHI agar and counted after 24 h.Following centrifugation at 1500 g for 12 min and passing through a 0.45 mm filter, wCD27 was enumerated by overlay plaque assay (in triplicate) with C. difficile NCTC 11204.
Total RNA extraction and cDNA production to demonstrate expression of putative phage repressors.The SV Total RNA Isolation System (Promega) was used for RNA extractions using the protocol for Gram-positive organisms.A total of 4.5 ml of each culture was extracted and RNA was eluted in 100 ml of nuclease-free water.Eluates were used immediately or flash-frozen in liquid nitrogen before being stored at 280 uC.The DNase step included in the SV Total RNA Isolation System was not sufficient to remove all traces of contaminating DNA.Eluates were therefore treated with TURBO DNase (Invitrogen) according to the manufacturer's instructions.In order to verify that all DNA had been eliminated, 1 ml was used as a template for PCR with Hotmaster Taq (5 Prime) using primers targeting the 16S rDNA V3 region (Table 1).Superscript III Reverse Transcriptase (Invitrogen) was used to produce cDNA from extracted RNA according to the manufacturer's instructions.

Determination of the phage integration site in NCTC 12727
Phage wCD27 can form lysogens in NCTC 12727; however its site of integration in the genome of this strain has not been determined.Typically phages integrate into the genome of their host by recombination between attP (on the phage) and target sequences in the bacteria termed attB.The attP site of most phages is immediately downstream of the integrase, which is predicted to be ORF42 in wCD27 (Mayer et al., 2008 and Fig. 1).Using a PCR based strategy (see Methods) we showed that wCD27 inserts into two specific sites in the C. difficile NCTC 12727 genome via recombination at the predicted attP site (Figs 1 and 2).The phage can also excise from this site, regenerating the attB and attP sites and generating a circular phage genome (Figs 1  and 2).In agreement with the latter observation, low levels of phage were detected in supernatants of uninduced cultures of NCTC 12727 (Fig. 3).
The first phage integration site is the same as that used by prophage 2 in C. difficile 630.This is within an open reading frame homologous to CD630-2952, which encodes a putative ATPase of an ABC transporter (integration site 1).The second phage integration site in NCTC 12727 is within an open reading frame homologous to the putative fliI gene in strain 630 (CD630-0251), which encodes a putative ATPase of the flagella protein export apparatus (integration site 2).

Infection of C. difficile NCTC11209 and differential toxin production
wCD27 produces plaques on four sensitive strains -NCTC 11204, 11205, 11207 and 11209 (Mayer et al., 2008).Using material derived from a plate of 11209 infected with wCD27 to create a zone of confluent lysis, a stable lysogen of NCTC 11209 was obtained.In this host the insertion of the phage was only detected in the ATPase of the ABC transporter in the same site as seen in NCTC 12727 (Figs 1 and 2).The NCTC 11209 lysogen produced considerably less toxin in the culture supernatant than its wild-type counterpart (P5,0.05;Fig. 4).Toxin activity of bacterial lysates was not tested.

Circularized
Reverse transcription PCR was used to assess putative phage repressor gene expression in the phage free NCTC 11209 and its lysogen (Fig. 5).No PCR products were obtained for the phage gene targets in NCTC 11209; in its lysogen, expression of orf44 only was observed, pointing to orf44 as encoding a candidate wCD27 repressor of the lytic cycle.

DISCUSSION
In this work, we determined the sequence of the attP site used by phage wCD27 to integrate into the C. difficile genome.The phage integrates into two sites in the C. difficile chromosome via its attP region.One of these sites is also used in a different C. difficile strain (11 209).Both insertion sites are within genes encoding putative ATPases; one of which is from an ATP transporter and the other within the putative ATPase of the flagella protein export apparatus.The insertions are likely to inactivate the target genes as they are predicted to insert in the region of the gene encoding the Walker A box of the ATPase.
Like other C. difficile phage (Meessen-Pinard et al., 2012), wCD27 spontaneously excises from the host genome and releases lytic phage.This could represent a survival strategy for the phage as presumably the majority of phage replicate along with the host genome but a small subset of the phage population is continually exploring potential new hosts.
In this work we also identified a gene encoding a potential phage repressor protein that is expressed only in the phage lysogens.Little is known about how C. difficile phage regulate and maintain lysogeny although Govind et al. (2009) have identified a potential repressor expressed in the phage wCD119 that binds to targets in both the prophage and the PaLoc, causing a reduction in toxin production in lysogens.In this work we also observed a reduction in toxin product in the lysogen of NCTC 11209, demonstrating that there is cross-talk between mobile elements and the PaLoc of C. difficile.The phage repressor represents a potential target for genetic manipulation of such phage to generate obligate lytic phage, which can be used in phage therapy of C. difficile-induced disease.Understanding of phage control circuits will also provide a deeper understanding of gene regulation in this important pathogen.) and the grey dotted line depicts level of phage release (log p.f.u.ml "1 ).

Fig. 1 .
Fig. 1.Location of prophages in NCTC 12727 and amplification of phage:chromosome junctions.(a) Integration in an orf homologous to CD_2952 (integration site 1) and (b) integration in fliI homologue (integration site 2).Phage genes are represented by thick black arrows and bacterial genes by white arrows.The direction of transcription is shown by the arrow, binding sites of primers used in PCR amplification are shown by thin arrows with direction of priming shown by the point of the arrow, and primer sequences are shown in Table 1.The dotted line represents the rest of the phage genome.The sequences at the phage:bacterial DNA junctions are shown in Fig. 2.Not drawn to scale.(c, d) Amplification of the phage:bacterial DNA junctions at integration site 1 (c) and integration site 2 (d) in NCTC 12727.Lane 1, LH junction; lane 2, RH junction; M, markers.(e) Amplification of the circularized wCD27 molecule in NCTC 12727.M, marker.
Fig. 2. DNA sequences of wCD27 integration sites.Phage DNA is shown in italics and bacterial DNA in plain text.The overlap region is shown in bold.The 1 bp difference between the attB sites is underlined.

Fig. 4 .
Fig. 4. Differential toxin production (combined toxin A and B assay) in wild-type and lysogenic strains of C. difficile NCTC 11209.Values are means of five replicate cultures±SD.

Fig. 3 .
Fig. 3. Spontaneous phage release in an uninduced culture of NCTC 12727.The black line shows the growth of C. difficile (logc.f.u.ml "1 ) and the grey dotted line depicts level of phage release (log p.f.u.ml "1 ).

Table 1 .
Primers used in this study