| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
C2 and comparisons to CD119 and inducible prophages of CD630
1 National University of Singapore, Yong Loo Lin School of Medicine, Department of Microbiology, 5 Science Drive 2, #05-03, 117597, Singapore
2 Microbiology and Immunology, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, WA 6009, Australia
3 Division of Microbiology and Infectious Diseases, The Western Australian Centre for Pathology and Medical Research, Nedlands, WA 6009, Australia
Correspondence
Barbara J. Chang
bchang{at}cyllene.uwa.edu.au
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
C2, is reported. The genome is 56 538 bp and organized into 84 putative ORFs in six functional modules. The head and tail structural proteins showed similarities to that of C. difficile phage CD119 and Streptococcus pneumoniae phage EJ-1, respectively. Homologues of structural and replication proteins were found in prophages 1 and 2 of the sequenced C. difficile CD630 genome. A putative holin appears unique to the C. difficile phages and was functional when expressed in Escherichia coli. Nucleotide sequence comparisons of C2 to CD119 and the CD630 prophage sequences showed relatedness between C2 and the prophages, but less so to CD119. C2 integrated into a gene encoding a putative transcriptional regulator of the gntR family. C2, CD119 and CD630 prophage 1 genomes had a Cdu1-attP-integrase arrangement, suggesting that the pathogenicity locus (PaLoc) of C. difficile, flanked by cdu1, has phage origins. The attP sequences of C2, CD119 and CD630 prophages were dissimilar. C2-related sequences were found in 84 % of 37 clinical C. difficile isolates and typed reference strains.
The GenBank accession number for the sequence reported in this paper is DQ466086.
A supplementary table and figure are available with the online version of this paper.
Present address: Department of Cell and Molecular Biology, Programme for Genomics and Bioinformatics, Karolinska Institutet, Berzelius väg 35, SE-171 77 Stockholm, Sweden.
Present address: School of Arts and Sciences, Monash University Malaysia, No. 2 Jalan Kolej, Bandar Sunway, Petaling Jaya 46150, Selangor Darul Ehsan, Malaysia.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) and Canada (Loo et al., 2005; Warny et al., 2005). The major virulence factors of C. difficile are toxins A and B encoded by tcdA and tcdB respectively, which are located on a 19 kb genomic region termed the pathogenicity locus (PaLoc) (Braun et al., 1996; Hammond & Johnson, 1995). The toxins are positively regulated by TcdR (Mani & Dupuy, 2001; Rupnik et al., 2005) and are negatively regulated by TcdC (Hundsberger et al., 1997; Matamouros et al., 2006); they are encoded by tcdR and tcdC respectively, also on the PaLoc. Another toxin-associated gene, tcdE, appears phage related but its function is unknown (Tan et al., 2001). C. difficile acquires antibiotic resistance and virulence genes through plasmids and transposons (Bruggemann, 2005) shown to significantly contribute to genome plasticity (Sebaihia et al., 2006). It is possible that phages also contribute to variance in virulence-associated genes (Lemee et al., 2005) and to the emergence of outbreak strains (McDonald et al., 2005). However, the prevalence of phage genes within C. difficile genomes is not known. In comparison to phages of Escherichia coli, Staphylococcus aureus and Lactobacillus species, the study of clostridial phages is in its infancy. Only one phage specific for C. difficile, temperate phage CD119, has been sequenced (Govind et al., 2006), while two putative prophage sequences were detected in the recently sequenced genome of C. difficile CD630 (Sebaihia et al., 2006).
Previously, we induced a temperate phage, C2, from a clinical C. difficile isolate. The phage was partially sequenced and characterized (Goh et al., 2005b) and was shown to increase toxin B levels in lysogens (Goh et al., 2005a). Two other induced temperate phages, C6 and C8, were also shown to have this effect in C. difficile lysogens (Goh et al., 2005a). While C. difficile toxin production itself was not phage mediated, phages may have some other role in host physiology. In this study, we compared the C2 genome with the genomes of CD119 and C. difficile CD630 (Sebaihia et al., 2006), as well as the unfinished sequence of C. difficile QCD-32g58. We found C2 genes to be prevalent in most of the clinical C. difficile isolates tested. Two phages, designated C630-1 and C630-2, were induced from CD630 in the course of this study.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Reference strains used were CCUG 37782, CCUG 20309 and CCUG 16126, which were purchased from the Culture Collection, University of Göteborg, Sweden. VPI 10463 was generously provided by Dr M. Rupnik, University of Ljubljana, Slovenia, and CD630 was kindly provided by Dr P. Mullany, University College London, UK. For induction of temperate phage, an overnight culture of CD630 in Brain Heart Infusion broth (BHIB, Oxoid) supplemented with 5 % horse blood, 50 µg erythromycin ml–1 (Sigma) and 10 µg tetracycline ml–1 (Sigma) was induced with 3 µg mitomycin C ml–1 (Sigma) and incubated for another 8 h. The culture supernatant was filtered through a 2 µm membrane (Pall), then assayed for phage against 10 randomly chosen clinical C. difficile strains (Table 1) as previously described (Goh et al., 2005b). Propagation of C630-1 and C630-2 with a 4 h culture of CD843 was as previously described for C6 (Goh et al., 2005b). C2 was propagated on CD062 as previously described (Goh et al., 2005b). C. difficile strains were maintained in Cooked Meat Medium (Oxoid), from which 37 °C overnight cultures in BHIB were prepared. Exponential-phase cultures were prepared by subculturing 1 ml of an overnight culture in 9 ml BHIB and incubating at 37 °C for 4 h.
|
). Phage DNA was also extracted directly from crude lysate using the Qiagen Lambda Midi Kit, according to the manufacturer's instructions. C2 DNA was digested with AccI, HincII, HindIII or XbaI and cloned into pUC19. Positive clones were selected for by blue–white colony screening, PCR using M13 primers and restriction enzyme digestion of recombinant plasmids. Sequencing was performed by the dideoxy chain-termination method with an automated ABI Prism 3100 DNA Sequencer (Applied Biosystems). Primers were designed and sequences were assembled using the Lasergene version 5.05 software (DNASTAR). A mean coverage of 3.53x (293 sequencing runs) and a minimum of 2x coverage (at least once in each strand) were obtained from sequencing the phage library. Gaps between contigs of C2 were filled and ends of the genome were sequenced by primer walking using phage genomic DNA as template. Probable protein-encoding genes (ORFs) were predicted using GeneMark.hmm VIOLIN and GeneMark.hmm for prokaryotes version 2.4 programs (Besemer & Borodovsky, 1999). Predicted ORFs were searched for similarity to proteins in databases by BLASTP (Altschul et al., 1990). Nucleotide similarity between C2 and C. difficile CD630 or QCD-32g58 was detected by BLASTN (Altschul et al., 1990) at http://www.sanger.ac.uk/Projects/C_difficile/ and http://www.ncbi.nlm.nih.gov/genomes/geblast.cgi?gi=5410, respectively. Alignments between two sequences were carried out with bl2seq (Tatusova & Madden, 1999) and the genome was searched for tRNA genes using the tRNAscan web server (Lowe & Eddy, 1997). Cumulative GC skew was carried out with GenSkew at http://mips.gsf.de/services/analysis/genskew, and a hydrophobicity plot was generated using Hydrophobicity grapher http://athena.bioc.uvic.ca/techDoc/hydrophobicity/ using the Kyte–Doolittle scaling system (Kyte & Doolittle, 1982). Transmembrane regions and beta-turns in an ORF were predicted by TMPRED at http://www.ch.embnet.org/software/TMPRED_form.html and BTPRED (Shepherd et al., 1999), respectively. Tandem Repeats Finder (Benson, 1999) was used to detect direct or inverted repeats in the genome.
Protein expression.
Plasmid pQE-hol and pQE-AbiF contained C2 putative ORF 36 and ORF 37, respectively, cloned between the BamHI and PstI sites of the pQE-30 expression vector (Qiagen). ORF 36 was amplified with Vent polymerase (NEB) using HolFBam (5'-CGCGGATCCATGGATAATTTAATAAG-3')/HolRPst (5'-AACTGCAGTTACTTTTCACCATCCT-3') with cycling conditions of 95 °C for 4 min, 30 cycles of 95 °C for 30 s, 53 °C for 30 s, 72 °C for 1 min and 72 °C for 10 min. ORF37 was amplified with Vent polymerase (NEB) and AbiFBam (5'-CGCGGATCCATGGTTGAAGTAAAAGA-3')/AbiFRPst (5'-AACTGCAGTTATTTAGCCAATATCTC-3') primers with cycling conditions of 95 °C for 2 min, 30 cycles of 95 °C for 30 s, 58 °C for 30 s, 72 °C for 2 min and 72 °C for 10 min. The PCR product was digested with BamHI and PstI and ligated into pQE-30 with complementary ends, then transformed into M15[pREP4] by electroporation (Bio-Rad Gene Pulser II). Recombinant pUC19 (NEB) plasmids harbouring C2 DNA inserts were grown at 37 °C in LB (Invitrogen) supplemented with 100 µg ampicillin ml–1 (ICN Biomedicals), 62 ng X-Gal ml–1 (Bio-Rad) and 0.625 mM IPTG (Sigma). Recombinant pQE-30 (Qiagen) expression plasmids were grown at 37 °C in LB supplemented with 100 µg ampicillin ml–1 and 25 µg kanamycin ml–1 (Sigma). Protein expression in M15[pREP4] cells was induced with 1 mM IPTG when growth of culture at 37 °C with shaking at 250 r.p.m. had reached an OD600 of 0.6.
Southern hybridization and dot blot.
C2 genomic probe was prepared by DIG-labelling of HincII-digested phage DNA according to the DIG High Prime DNA Labelling and Detection Starter Kit 1 (Roche) instructions. C. difficile and phage DNA were digested by HincII or HindIII and XbaI, separated in 1 % TBE agarose and transferred to a nylon membrane (Amersham) as previously described (Sambrook et al., 1989). Hybridization was carried out at 37–39 °C for 16–20 h, followed by stringent washing and colour development of membranes as in the system manual. Dot blots were carried out by spotting 120–200 ng chromosomal DNA on nylon membranes, fixing the DNA by microwave on high power for 2.5 min (Angeletti et al., 1995) followed by hybridization to C2 genomic probe, according to the DIG system manual (Roche).
CHEF electrophoresis.
Undigested phage DNA (150–200 ng) was added to an equal volume of molten 1 % low-melting-point Agarose (Sigma), then loaded into a 1 % TBE Pulse Field Certified Agarose (Bio-Rad). Electrophoresis was carried out in 0.5x TBE running buffer using the CHEF-DR II Pulse Field Electrophoresis System (Bio-Rad) at pulse times of 3–13 s, 200 V for 22 h.
SDS-PAGE and N-terminal sequencing.
SDS-PAGE analysis of phage was carried out as previously described (Ford et al., 1998) with modifications. C2 was purified through a CsCl density gradient as previously described (Goh et al., 2005b); 20 µl (107 p.f.u. ml–1) was repeatedly frozen in liquid nitrogen and thawed at 37 °C three times, heated at 75 °C for 4 min, mixed with 5x sample buffer (0.255 M Tris pH 6.8, 50 %, v/v, glycerol, 5 % w/v SDS, 0.05 %, w/v, bromophenol blue, 0.25 M DTT) and heated again at 95 °C for 5 min before electrophoresis. Electrophoresis was carried out as previously described (Laemmli, 1970) using the Mini-PROTEAN II cell (Bio-Rad) and 12 % acrylamide gels, which were stained with BLUPRINT Fast-PAGE Stain (Gibco BRL). Proteins were electroblotted to PVDF membranes (Bio-Rad) as described by Sambrook et al. (1989) using cold tank blotting transfer buffer (25 mM Tris pH 8.3, 150 mM glycine, 20 % v/v methanol). The PVDF membrane was stained with Coomassie blue R250 and two major bands were excised from the membrane. Ten amino acids from the N-terminus of each protein were determined by an automated sequencer (Applied Biosystems 477 Protein Sequencer).
Determination of the attP region.
The attP region was predicted to be downstream of the integrase, and primer pair patt-1/int-1 was used to generate a 311 bp PCR product which was DIG labelled and used as an attP probe for hybridization at 39 °C. Primer sequences are: patt-1, 5'-GTAAAGATGATGAAGTGGATGAAG-3'; int-1, 5'-GCATTTTACAATAATTTGCCACCG-3'. The cycling conditions were 95 °C for 5 min, 30 cycles of 95 °C for 30 s, 50 °C for 30 s, 72 °C for 1 min and 72 °C for 10 min. Genomic DNA of CD242 (5 µg) was digested with AccI, HincII or HindIII and self-ligated with T4 DNA ligase. Inverse PCR (Ochman et al., 1988) was carried out with divergent primer pairs patt-0/Cdu1-1 and int-3/int-5. Primer sequences were as follows: patt-0, 5'-CTGTGAATGTAGTTGATTCATTC-3'; Cdu1-1, 5'-GGTTAGAGCGAAGGGAGTTT-3'; int-3, 5'-CAACCACTATGGACACATATTC-3'; int-5, 5'-CGCAAGCAATGAAAATTAAAG-3'. The PCR products were sequenced with the same primers. To determine attBP', an additional primer, gntR-F (5'-GGATTTAGAAGTAAATTCC-3') was used.
Electron microscopy.
Carbon-coated copper Formvar grids (400 mesh, SPI supplies) were floated on 20 µl 0.01 % poly-L-lysine for 30 s and excess liquid was wicked off with a filter paper. The grid was floated on 20 µl phage suspension (107–109 p.f.u ml–1) for 3 min and dried as above, then fixed with 20 µl 1 % glutaraldehyde for 1 min and negatively stained with 1 % phosphotungstic acid pH 7.4 for 30–90 s. The grids were air-dried for 30 min and viewed under a Philips 2085 transmission electron microscope at 57 000–89 000x magnification, operating at 100 kV. Dimensions of a minimum of four phage particles for each phage were measured, and results expressed as mean±SD.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
C2 genome) while the unit genome length was 56 538 bp as determined by sequencing, indicating terminal redundancy of approximately 3.2 kb in the packaged genome. The G+C content was 28.72 mol%, which is identical to the recently sequenced CD119 (Govind et al., 2006) and slightly lower than that of the C. difficile CD630 genome (29.06 %). A total of 84 putative ORFs were identified, of which seven (8 %) showed no homology to proteins in the NCBI and EBI databases; 37 ORFs (44 %) had sequence similarity to proteins with unknown functions, of which 13 ORFs (15 %) were found only in C. difficile phage/prophage genomes. A supplementary table detailing the predicted ORFs and similarities to other proteins is included with the online version of this paper. In general, the predicted ORFs in the left and right arms of the genome had sequence similarity to CD119 and CD630 prophages 1 and 2, while ORFs in the middle section were similar to various bacterial or phage proteins. No programmed frameshift signals for translation were detected. The proteins of C2 were separated by 12 % SDS-PAGE (Fig. 1b) and two major proteins of C2 were identified as products of ORF 7, a putative capsid, and ORF 13, a putative tail sheath with the N-terminal methionine removed as expected (Ben-Bassat et al., 1987; Romero et al., 2004). Cumulative GC skew analysis of the genome sequence, included as a supplementary figure with the online version of this paper, revealed a putative origin of replication in the region of nt 37929 and a putative terminus of replication in the region of nt 30390.
|
C2 genome). There was consistently high homology to ORFs of CD630 prophage 1 and 2, while sequence similarity to CD119 was sporadic. Of interest within the tail assembly module is a cassette of genes found in phage EJ-1 of Streptococcus pneumoniae (Romero et al., 2004). The cell lysis module consisting of ORFs 36, 37 and 38 was atypical in the relative positions of the putative endolysin and holin, which are usually adjacent to each other (Young, 1992). Putative holins of the C. difficile phages had high sequence similarity to one another and good alignment with holin of another phage, despite low sequence homology (Fig. 3). A hydrophobicity plot (not shown) of ORF 36 revealed transmembrane regions separated by 
-turns and outside N- and C-termini typical of type II holins (Wang et al., 2000). Constitutive expression of ORF 36 inhibited growth and induced expression of ORF 36 decreased turbidity in E. coli, while expression of ORF 37 did not have an effect on cell growth (Fig. 4). Similar results have been shown for other phage holins (Muyombwe et al., 1999; Sheehan et al., 1999) and for C. difficile tcdE (Tan et al., 2001), which had good sequence alignment with holins (Fig. 3).
|
|
|
). These enzymes, together with a centromere-like sequence, parS, are required for maintenance of DNA stability (Austin & Abeles, 1983; Yamaichi & Niki, 2000); therefore ORFs 41 and 42 could be part of the lysogeny module. Possible parS sequences in the form of short direct or inverted repeats (Dam & Gerdes, 1994; Gallie & Kado, 1987; Radnedge et al., 1996) around ORFs 41 and 42 were not detected, as was the case for LE1 (Bourhy et al., 2005). Sequence similarities of ORF 41 to TcdB (22 % of 279 amino acids, E-value 1.1) and ORF 46 to Cdu1 (29 % of 125 amino acids, E-value 4x10–12) are noteworthy. The C2 lysogeny module is unusual in being extended compared to that of 
(Birge, 2000); there appear to be two sets of repressors (ORFs 49, 50, 58 and ORFs 51, 53, 61) and perhaps as a consequence, two antirepressors (ORFs 52 and 55). ORF 59 had low percentage sequence similarity to an excisionase, the first to be detected in C. difficile phages. Although not shown, homologues of ORFs 10–14, 16, 20–22 and 24–26 have also been detected in the Clostridium sp. strain OhILAs sequence with high percentage sequence similarity. The attP of C2 in CD242 was predicted to be between ORF 46 (transcriptional regulator/Cdu1 homologue) and ORF 47 (integrase), and was confirmed by Southern hybridization (Fig. 5a). The attP region was flanked by AccI, HincII and HindIII sites; digestion of lysogenic DNA with either enzyme was used for inverse PCR; however, only an attPB' PCR product was obtained (Fig. 5b, c). As the bacterial sequence of attPB' had high percentage similarity to a gntR transcriptional regulator in CD630 (92 % of 93 amino acids, E-value 1.4x10–38) and QCD-32g58, a forward primer specific for gntR (gntR-F) and int-3 were used to determine the sequence of attBP' (Fig. 5c). The C2 attachment site sequence CTGTGAGAAAT is different from that of CD119 (TTTATATGTGTTAT), CD630 prophage 1 (TAAAGATGA) and prophage 2 (TCCACTAGG). Interestingly, the translated 3' end of the integrated CD630 prophage 1 (nt 1143302–1143688) was similar to Cdu1 (36 % of 125 amino acids, E-value 3x10–11) and attP of CD119 was 248 nt downstream of a Cdu1 homologue (Govind et al., 2006).
|
CD119 and prophage 1 and 2 homologues were mostly found in the DNA replication, recombination and modification module of the C2 genome. ORF 65, common to the C. difficile phages, is likely an essential recombination function (Erf) protein that is a member of a superfamily of single-strand annealing proteins involved in phage genome circularization via homologous recombination following DNA entry (Iyer et al., 2002). Also common was ORF 78, with high sequence similarity to RusA, an enzyme thought to have phage origins (Sharples et al., 2002). RusA is a DNA endonuclease that resolves Holliday junctions in DNA replication, recombination and repair (Mahdi et al., 1996).
Relatedness of C2, C630-1, C630-2 and CD119 and prevalence of prophage genes in C. difficile isolates
Mitomycin C induction of CD630 resulted in two plaque types which corresponded to phage particles of the same morphology but having slightly different head sizes. Since the genome of prophage 1 (55 850 bp) in CD630 is larger than prophage 2 (49 178 bp), it is likely that prophage 1 produced larger particles (C630-1) compared to prophage 2 (C630-2). C630-1 particles measured 31.7±0.7 nm in head diameter and 62.4±5.1 nm in tail length, while C630-2 particle dimensions were 28.1±1.3 nm (head) and 39.5±5.8 nm (tail) (Fig. 6). BLASTN identified regions of nucleotide sequence similarity throughout the C2 genome to regions of CD630 that indicated the C630-1 and C630-2 prophages have similar genome organization (Fig. 2
). Dot plots (not shown) of C2 and the hypothetical genome sequences of C630-1 and C630-2 were virtually identical. Hence, common frameshift regions in C2 were detected at nt 26290–26996, nt 32933–33469, nt 47350–50177 (C630-1) and nt 42546–53713 (C630-2). Nucleotide comparison of C2 and CD119 genomes by dot plot (not shown) revealed some regions of similarity (Fig. 2) but were a less similar pair compared to C2/C630-1 and C2/C630-2. Dot-plot comparisons of CD119 to C630-1 and C630-2 revealed sequence similarity in parts of the cell lysis, lysogeny control and DNA replication and modification modules of the three phages.
|
C2-related genes in clinical C. difficile strains, dot blots and Southern blots were carried out with a C2 genomic probe. The results showed that out of 37 strains tested (Table 1), only six (CD13, CD57, CD62, CD062, CD843 and CCUG 37782) did not contain prophage genes; 31 strains including four typed reference strains exhibited homology to varying degrees. Differences in dot-blot signals were further analysed by Southern hybridization (not shown). The most common homologous bands found in almost all strains correlated to ORFs 7–13 of the head structural module and ORFs 78–84 of the DNA replication, recombination and modification module, respectively. Regions representing modules of lysogeny control, lysis, head and DNA methylase were also found in some strains. To determine whether C2-related phage genes were present in another sequenced C. difficile strain, QCD-32g58, a BLASTN search was carried out. Only three regions of high sequence similarity were found between QCD-32g58 and C2 (Fig. 2). However, a whole prophage genome was not detected in QCD-32g58. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Juhala et al., 2000) and co-evolution of bacterial hosts and phage (Kwan et al., 2005; Pedulla et al., 2003). An aim of this study was to compare the genome of our previously partially characterized phage C2 to other phage genomes to provide an insight into phages of C. difficile.
The genome organization of C2 is typical of phages with a lysogenic cycle (Canchaya et al., 2003). Its genome length was previously underestimated by addition of restriction fragments separated under normal electrophoresis (Goh et al., 2005b). It was previously shown not to possess cohesive ends (Goh et al., 2005b) and genome sequencing revealed it to have terminally redundant ends. The packaging mechanism of a terminase may be predicted from the large subunit amino acid sequence (Casjens et al., 2005); the C2 terminase large subunit had high percentage sequence similarity to SPP1 (26 % of 426 amino acids, E-value 3x10–25) of the P22-like headful subgroup. This suggests the ends of C2 were likely to be generated by a headful packaging mechanism. Based on the number of protein homologues and unique hypothetical proteins, C2 is closely related to the other C. difficile phages, CD119, C630-1 and C630-2, and demonstrates modular mosaicism (Casjens, 2005). Gene divergence appears greatest within the lysogeny control module, followed by tail structural proteins, which is common for tailed temperate phages as a method of diversifying infectable hosts (Casjens, 2005). Tail structural components of C2 may have been derived from an ancestral phage of EJ-1, while a putative LysM within tail-associated proteins may indicate lytic enzymes are used for local cell wall degradation and hence penetration of host wall for injection of phage DNA, similar to tailspike proteins of P22, Sf6 (Freiberg et al., 2003) and T4 (Kanamaru et al., 2005) and tail fibre proteins of anti-K1 phages (Muhlenhoff et al., 2003). Homologues of extrachromosomal replicative proteins ParA and ParB found close to the lysogeny module and their relative direction of transcription suggest their expression is associated with lysogenic conversion. Interesting possibilities include C2 switching to an LE1-like replicative prophage state for stability and perhaps having a pseudolysogeny phase (i.e. genome does not integrate into host chromosome but rather exists as a circular intermediate), or that it was once a replicative prophage. Low percentage sequence homology between ORF 41 and TcdB suggests phage origins of the toxin and may explain the genetic variability in tcdB, which has been observed in C. difficile toxinotypes (Rupnik et al., 2001), more commonly in tcdB than tcdA (Rupnik et al., 1998). Sequence similarity of ORF 46 to Cdu1, which borders the PaLoc, and the holin having a similar effect on E. coli as TcdE also point toward the PaLoc as a collection of genes transferred by phages, which have evolved to become the current virulence genes of C. difficile. ORF 46 (Cdu1 homologue) and its downstream non-coding region appear to be involved in integration because the Cdu1-attP-integrase arrangement is conserved in C2, C630-1 and CD119. The attPs of C2 and CD119 are 197 nt and 248 nt downstream of a Cdu1 homologue (Govind et al., 2006), respectively, while cdu1 contains an attP for C630-1 (Sebaihia et al., 2006). There does not appear to be a preferred site for the integration of this group of phages in C. difficile, as the attachment sites are different for each phage. The presence of an AbiF protein on the phage genome is unusual; it is normally carried on a plasmid and confers phage resistance to bacteria, resulting in an abortive phage infection at the level of phage DNA replication (Garvey et al., 1995). The advantage of carrying abiF is unknown and has not been found on other phage genomes; one possibility could be to prevent superinfection of C2 lysogens by unrelated phages that are susceptible to AbiF.
Comparative DNA sequence analysis of C2, C630-1, C630-2 and CD119 showed the degree of pairwise relatedness to be C630-1/C2>C2/C630-2>C630-1/CD119>CD119/C630-2>CD119/C2. This indicates that C630-1 and C630-2 are intermediates of C2 and CD119 in the evolutionary sense. The brief sampling of randomly chosen clinical C. difficile isolates revealed that C2-related prophage genes are prevalent in C. difficile. Although these may represent only phage remnants, the apparently low prevalence of inducible phage (Goh et al., 2005b) may simply be due to the lack of appropriate indicator strains. Isolates possessing both tcdA and tcdB (e.g. CD61) were as likely to be devoid of C2-related genes as isolates possessing truncated versions of tcdA and/or tcdB (e.g. CCUG 20309, CD843), or lacking either or both toxin genes (CCUG 37782, CD55). In general, there was no correlation between the presence of tcdA and tcdB and prevalence of C2-related prophage genes in C. difficile strains. Therefore, the current role of C2 is not in generating genetic diversity within the PaLoc but perhaps in other areas of the host genome related to virulence. The following ORFs may affect host fitness: ORF 19 (putative TerD), ORF 9 (sequence similarity to the ‘Alkaliphilus metalliredigenes’ sigma-54 interaction region, 28 % of 114 amino acids, E-value 0.054) and ORF 15 (low sequence similarity to Clostridium thermocellum S-layer protein). C2 disruption of a gntR-like transcriptional regulator potentially affects expression of host genes, which may lead to altered fitness. In CD630, gntR was upstream of genes encoding the mannose-specific phosphotransferase system (PTS) (Sebaihia et al., 2006). The mannose PTS is involved in sugar transport and global regulation of gene expression, in a number of Gram-positive genera (Abranches et al., 2003; Arous et al., 2004; Chaillou et al., 2001; Reizer et al., 1999), including the regulation of energy metabolism and virulence genes in Streptococcus mutans (Abranches et al., 2006). Hypothetically, integration of C2 into CD630 could lead to significant changes in the C. difficile phenotype through mannose PTS deregulation. The contribution of C2 and related temperate phages to the physiology of C. difficile and their potential roles in gene transfer and as genetic tools for this species are worthy of further investigation.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: P. R. Herron
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Abranches, J., Candella, M. M., Wen, Z. T., Baker, H. V. & Burne, R. A. (2006). Different roles of EIIABMan and EIIGlc in regulation of energy metabolism, biofilm development, and competence in Streptococcus mutans. J Bacteriol 188, 3748–3756.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef][Medline]
Angeletti, B., Battiloro, E., Pascale, E. & D'Ambrosio, E. (1995). Southern and Northern blot fixing by microwave oven. Nucleic Acids Res 23, 879–880.
Arous, S., Dalet, K. & Hechard, Y. (2004). Involvement of the mpo operon in resistance to class IIa bacteriocins in Listeria monocytogenes. FEMS Microbiol Lett 238, 37–41.[Medline]
Austin, S. & Abeles, A. (1983). Partition of unit-copy miniplasmids to daughter cells. II. The partition region of miniplasmid P1 encodes an essential protein and a centromere-like site at which it acts. J Mol Biol 169, 373–387.[CrossRef][Medline]
Barbut, F., Mario, N., Delmee, M., Gozian, J. & Petit, J. C. (1993). Genomic fingerprinting of Clostridium difficile isolates by using a random amplified polymorphic DNA (RAPD) assay. FEMS Microbiol Lett 114, 161–166.[CrossRef][Medline]
Ben-Bassat, A., Bauer, K., Chang, S. Y., Myambo, K., Boosman, A. & Chang, S. (1987). Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure. J Bacteriol 169, 751–757.
Benson, G. (1999). Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res 27, 573–580.
Besemer, J. & Borodovsky, M. (1999). Heuristic approach to deriving models for gene finding. Nucleic Acids Res 27, 3911–3920.
Birge, E. A. (2000). Bacterial and Bacteriophage Genetics, 4th edn. New York: Springer.
Bourhy, P., Frangeul, L., Couve, E., Glaser, P., Saint Girons, I. & Picardeau, M. (2005). Complete nucleotide sequence of the LE1 prophage from the spirochete Leptospira biflexa and characterization of its replication and partition functions. J Bacteriol 187, 3931–3940.
Braun, V., Hundsberger, T., Leukel, P., Sauerborn, M. & von Eichel-Streiber, C. (1996). Definition of the single integration site of the pathogenicity locus in Clostridium difficile. Gene 181, 29–38.[CrossRef][Medline]
Bruggemann, H. (2005). Genomics of clostridial pathogens: implication of extrachromosomal elements in pathogenicity. Curr Opin Microbiol 8, 601–605.[CrossRef][Medline]
Canchaya, C., Proux, C., Fournous, G., Bruttin, A. & Brussow, H. (2003). Prophage genomics. Microbiol Mol Biol Rev 67, 238–276.
Casjens, S. R. (2005). Comparative genomics and evolution of the tailed-bacteriophages. Curr Opin Microbiol 8, 451–458.[CrossRef][Medline]
Casjens, S. R., Gilcrease, E. B., Winn-Stapley, D. A., Schicklmaier, P., Schmieger, H., Pedulla, M. L., Ford, M. E., Houtz, J. M., Hatfull, G. F. & Hendrix, R. W. (2005). The generalized transducing Salmonella bacteriophage ES18: complete genome sequence and DNA packaging strategy. J Bacteriol 187, 1091–1104.
Chaillou, S., Postma, P. W. & Pouwels, P. H. (2001). Contribution of the phosphoenolpyruvate : mannose phosphotransferase system to carbon catabolite repression in Lactobacillus pentosus. Microbiology 147, 671–679.
Dam, M. & Gerdes, K. (1994). Partitioning of plasmid R1. Ten direct repeats flanking the parA promoter constitute a centromere-like partition site parC, that expresses incompatibility. J Mol Biol 236, 1289–1298.[CrossRef][Medline]
Ford, M. E., Stenstrom, C., Hendrix, R. W. & Hatfull, G. F. (1998). Mycobacteriophage TM4: genome structure and gene expression. Tuber Lung Dis 79, 63–73.[CrossRef][Medline]
Freiberg, A., Morona, R., Van den Bosch, L., Jung, C., Behlke, J., Carlin, N., Seckler, R. & Baxa, U. (2003). The tailspike protein of Shigella phage Sf6. A structural homolog of Salmonella phage P22 tailspike protein without sequence similarity in the beta-helix domain. J Biol Chem 278, 1542–1548.
Gallie, D. R. & Kado, C. I. (1987). Agrobacterium tumefaciens pTAR parA promoter region involved in autoregulation, incompatibility and plasmid partitioning. J Mol Biol 193, 465–478.[CrossRef][Medline]
Garvey, P., Fitzgerald, G. F. & Hill, C. (1995). Cloning and DNA sequence analysis of two abortive infection phage resistance determinants from the lactococcal plasmid pNP40. Appl Environ Microbiol 61, 4321–4328.[Abstract]
Goh, S., Chang, B. J. & Riley, T. V. (2005a). Effect of phage infection on toxin production by Clostridium difficile. J Med Microbiol 54, 129–135.
Goh, S., Riley, T. V. & Chang, B. J. (2005b). Isolation and characterization of temperate bacteriophages of Clostridium difficile. Appl Environ Microbiol 71, 1079–1083.
Govind, R., Fralick, J. A. & Rolfe, R. D. (2006). Genomic organization and molecular characterization of Clostridium difficile bacteriophage CD119. J Bacteriol 188, 2568–2577.
Hammond, G. A. & Johnson, J. L. (1995). The toxigenic element of Clostridium difficile strain VPI 10463. Microb Pathog 19, 203–213.[CrossRef][Medline]
Haslam, S. C., Ketley, J. M., Mitchell, T. J., Stephen, J., Burdon, D. W. & Candy, D. C. (1986). Growth of Clostridium difficile and production of toxins A and B in complex and defined media. J Med Microbiol 21, 293–297.
Hendrix, R. W. (2002). Bacteriophages: evolution of the majority. Theor Popul Biol 61, 471–480.[CrossRef][Medline]
Hundsberger, T., Braun, V., Weidmann, M., Leukel, P., Sauerborn, M. & von Eichel-Streiber, C. (1997). Transcription analysis of the genes tcdA-E of the pathogenicity locus of Clostridium difficile. Eur J Biochem 244, 735–742.[Medline]
Iyer, L. M., Koonin, E. V. & Aravind, L. (2002). Classification and evolutionary history of the single-strand annealing proteins, RecT, Redbeta, ERF and RAD52. BMC Genomics 3, 8.[Medline]
Juhala, R. J., Ford, M. E., Duda, R. L., Youlton, A., Hatfull, G. F. & Hendrix, R. W. (2000). Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lambdoid bacteriophages. J Mol Biol 299, 27–51.[CrossRef][Medline]
Kanamaru, S., Ishiwata, Y., Suzuki, T., Rossmann, M. G. & Arisaka, F. (2005). Control of bacteriophage T4 tail lysozyme activity during the infection process. J Mol Biol 346, 1013–1020.[CrossRef][Medline]
Kwan, T., Liu, J., DuBow, M., Gros, P. & Pelletier, J. (2005). The complete genomes and proteomes of 27 Staphylococcus aureus bacteriophages. Proc Natl Acad Sci U S A 102, 5174–5179.
Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105–132.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Lemee, L., Bourgeois, I., Ruffin, E., Collignon, A., Lemeland, J. F. & Pons, J. L. (2005). Multilocus sequence analysis and comparative evolution of virulence-associated genes and housekeeping genes of Clostridium difficile. Microbiology 151, 3171–3180.
Loo, V. G., Poirier, L., Miller, M. A., Oughton, M., Libman, M. D., Michaud, S., Bourgault, A. M., Nguyen, T., Frenette, C. & other authors (2005). A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N Engl J Med 353, 2442–2449.
Lowe, T. M. & Eddy, S. R. (1997). tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25, 955–964.
Mahdi, A. A., Sharples, G. J., Mandal, T. N. & Lloyd, R. G. (1996). Holliday junction resolvases encoded by homologous rusA genes in Escherichia coli K-12 and phage 82. J Mol Biol 257, 561–573.[CrossRef][Medline]
Mani, N. & Dupuy, B. (2001). Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor. Proc Natl Acad Sci U S A 98, 5844–5849.
Matamouros, S., Govind, R. & Dupuy, B. (2006). TcdC inhibits toxin synthesis in Clostridium difficile. In 5th International Meeting on the Molecular Biology and Pathogenesis of the Clostridia, p. 64. Nottingham, UK.
McDonald, L. C., Killgore, G. E., Thompson, A., Owens, R. C., Jr, Kazakova, S. V., Sambol, S. P., Johnson, S. & Gerding, D. N. (2005). An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med 353, 2433–2441.
Muhlenhoff, M., Stummeyer, K., Grove, M., Sauerborn, M. & Gerardy-Schahn, R. (2003). Proteolytic processing and oligomerization of bacteriophage-derived endosialidases. J Biol Chem 278, 12634–12644.
Muyombwe, A., Tanji, Y. & Unno, H. (1999). Cloning and expression of a gene encoding the lytic functions of Bacillus amyloliquefaciens phage: evidence of an auxiliary lysis system. J Biosci Bioeng 88, 221–225.[CrossRef][Medline]
Ochman, H., Gerber, A. S. & Hartl, D. L. (1988). Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–623.
Pedulla, M. L., Ford, M. E., Houtz, J. M., Karthikeyan, T., Wadsworth, C., Lewis, J. A., Jacobs-Sera, D., Falbo, J., Gross, J. & other authors (2003). Origins of highly mosaic mycobacteriophage genomes. Cell 113, 171–182.[CrossRef][Medline]
Radnedge, L., Davis, M. A. & Austin, S. J. (1996). P1 and P7 plasmid partition: ParB protein bound to its partition site makes a separate discriminator contact with the DNA that determines species specificity. EMBO J 15, 1155–1162.[Medline]
Reizer, J., Bachem, S., Reizer, A., Arnaud, M., Saier, M. H., Jr & Stulke, J. (1999). Novel phosphotransferase system genes revealed by genome analysis – the complete complement of PTS proteins encoded within the genome of Bacillus subtilis. Microbiology 145, 3419–3429.
Romero, P., Lopez, R. & Garcia, E. (2004). Genomic organization and molecular analysis of the inducible prophage EJ-1, a mosaic myovirus from an atypical pneumococcus. Virology 322, 239–252.[CrossRef][Medline]
Rupnik, M., Avesani, V., Janc, M., von Eichel-Streiber, C. & Delmee, M. (1998). A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates. J Clin Microbiol 36, 2240–2247.
Rupnik, M., Brazier, J., Duerden, B., Grabnar, M. & Stubbs, S. (2001). Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains and description of novel toxinotypes. Microbiology 147, 439–447.
Rupnik, M., Dupuy, B., Fairweather, N. F., Gerding, D. N., Johnson, S., Just, I., Lyerly, D. M., Popoff, M. R., Rood, J. I. & other authors (2005). Revised nomenclature of Clostridium difficile toxins and associated genes. J Med Microbiol 54, 113–117.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sebaihia, M., Wren, B. W., Mullany, P., Fairweather, N. F., Minton, N., Stabler, R., Thomson, N. R., Roberts, A. P., Cerdeno-Tarraga, A. M. & other authors (2006). The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat Genet 38, 779–786.[CrossRef][Medline]
Sharples, G. J., Bolt, E. L. & Lloyd, R. G. (2002). RusA proteins from the extreme thermophile Aquifex aeolicus and lactococcal phage r1t resolve Holliday junctions. Mol Microbiol 44, 549–559.[CrossRef][Medline]
Sheehan, M. M., Stanley, E., Fitzgerald, G. F. & van Sinderen, D. (1999). Identification and characterization of a lysis module present in a large proportion of bacteriophages infecting Streptococcus thermophilus. Appl Environ Microbiol 65, 569–577.
Shepherd, A. J., Gorse, D. & Thornton, J. M. (1999). Prediction of the location and type of beta-turns in proteins using neural networks. Protein Sci 8, 1045–1055.[Medline]
Tan, K. S., Wee, B. Y. & Song, K. P. (2001). Evidence for holin function of tcdE gene in the pathogenicity of Clostridium difficile. J Med Microbiol 50, 613–619.
Tatusova, T. A. & Madden, T. L. (1999). BLAST 2 sequences, a new tool for comparing protein and nucleotide sequences. FEMS Microbiol Lett 174, 247–250.[CrossRef][Medline]
Wang, I. N., Smith, D. L. & Young, R. (2000). Holins: the protein clocks of bacteriophage infections. Annu Rev Microbiol 54, 799–825.[CrossRef][Medline]
Warny, M., Pepin, J., Fang, A., Killgore, G., Thompson, A., Brazier, J., Frost, E. & McDonald, L. C. (2005). Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet 366, 1079–1084.[CrossRef][Medline]
Yamaichi, Y. & Niki, H. (2000). Active segregation by the Bacillus subtilis partitioning system in Escherichia coli. Proc Natl Acad Sci U S A 97, 14656–14661.
Young, R. (1992). Bacteriophage lysis: mechanism and regulation. Microbiol Rev 56, 430–481.
Received 13 September 2006;
revised 19 November 2006;
accepted 27 November 2006.
This article has been cited by other articles:
|
|
 |
|
  R. Govind, G. Vediyappan, R. D. Rolfe, B. Dupuy, and J. A. Fralick Bacteriophage-Mediated Toxin Gene Regulation in Clostridium difficile J. Virol., December 1, 2009; 83(23): 12037 - 12045. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Mayer, A. Narbad, and M. J. Gasson Molecular Characterization of a Clostridium difficile Bacteriophage and Its Cloned Biologically Active Endolysin J. Bacteriol., October 15, 2008; 190(20): 6734 - 6740. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L.-C. Fortier and S. Moineau Morphological and Genetic Diversity of Temperate Phages in Clostridium difficile Appl. Envir. Microbiol., November 15, 2007; 73(22): 7358 - 7366. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
) up to 4 h post-induction. A similar trend was observed for tcdE expression (pQE-tcdE, 
). Expression of another phage gene (abiF) did not result in cell growth retardation (pQE-abiF, 
).
