Morphological and genetic diversity of temperate phages in Clostridium difficile

Eight temperate phages were characterized after mitomycin C induction of six Clostridium difficile isolates corresponding to six distinct PCR ribotypes. The hypervirulent C. difficile strain responsible for a multi-institutional outbreak (NAP1/027 or QCD-32g58) was among these prophage-containing strains. Observation of the crude lysates by transmission electron microscopy (TEM) revealed the presence of three phages with isometric capsids and long contractile tails (Myoviridae family), as well as five phages with long noncontractile tails (Siphoviridae family). TEM analyses also revealed the presence of a significant number of phage tail-like particles in all the lysates. Southern hybridization experiments with restricted prophage DNA showed that C. difficile phages belonging to the family Myoviridae are highly similar and most likely related to previously described prophages phiC2, phiC5, and phiCD119. On the other hand, members of the Siphoviridae phage family are more genetically divergent, suggesting that they originated from distantly related ancestors. Our data thus suggest that there are at least three genetically distinct groups of temperate phages in C. difficile; one group is composed of highly related myophages, and the other two groups are composed of more genetically heterogeneous siphophages. Finally, no gene homologous to genes encoding C. difficile toxins or toxin regulators could be identified in the genomes of these phages using DNA hybridization. Interestingly, each unique phage restriction profile correlated with a specific C. difficile PCR ribotype.     

Physical mapping of Streptomyces coelicolor A3(2). V. Structural modifications of actinophage phiC43 DNA molecules

As shown by genetical and physical methods, the total preparation of phiC43 phage obtained after spontaneous induction of the prophage from S. lividans 803 strain is a heterogenous population. The wild-type phage (phi C43 wt) is only represented in 5--10% of the population. The majority of phage variants are not able to establish the lysogenic state. The structure of DNA molecules of some phages from the total preparations was characterized by electron microscopy of DNA heteroduplexes. Molecules of phiC43 wt DNA appeared to be completely homologous to those of recently studied phiC62 phage, except for two small regions of approximately 0.3 kb in the central part. Phage variants defective in establishment of the lysogenic state were distributed to two groups. One of them consists of deletion variants, the other--deletion/insertion variants. Deletions in DNA molecule of all nonlysogenizing phage overlap. The region of overlapping seems to be responsible for establishment of the lysogenic state. In the same region, deletion of DNA molecules of mutant phiC311 yg2 has been located. Three deletion/insertion variants contain homologous foreign sequences of various length. It is likely that these insertions are fragments of the host chromosomal DNA.     

Clostridium sordellii bacteriophage active on Clostridium difficile

Among five strains of Clostridium difficile and 39 strains of Cl. sordellii tested, one Cl. difficile phage and four Cl. sordellii phages were found to be lytic for Cl. difficille strain 2. The five phages were similar in morphology, showing a polyhedral head of 60 nm in diameter, a tail of 105–120 nm, a contractile tail sheath and a base plate. They were sensitive to heat (60°C/10 min) and stable at 4°C for at least 6 months. As the phage donor strains and the indicator strain were not cytotoxigenic, no phage-infected culture of Cl. difficile 2 was able to produce cytotoxin.     

Prophage Carriage and Diversity within Clinically Relevant Strains of Clostridium difficile

Prophages are encoded in most genomes of sequenced Clostridium difficile strains. They are key components of the mobile genetic elements and, as such, are likely to influence the biology of their host strains. The majority of these phages are not amenable to propagation, and therefore the development of a molecular marker is a useful tool with which to establish the extent and diversity of C. difficile prophage carriage within clinical strains. To design markers, several candidate genes were analyzed including structural and holin genes. The holin gene is the only gene present in all sequenced phage genomes, conserved at both terminals, with a variable mid-section. This allowed us to design two sets of degenerate PCR primers specific to C. difficile myoviruses and siphoviruses. Subsequent PCR analysis of 16 clinical C. difficile ribotypes showed that 15 of them are myovirus positive, and 2 of them are also siphovirus positive. Antibiotic induction and transmission electron microscope analysis confirmed the molecular prediction of myoviruses and/or siphovirus presence. Phylogenetic analysis of the holin sequences identified three groups of C. difficile phages, two within the myoviruses and a divergent siphovirus group. The marker also produced tight groups within temperate phages that infect other taxa, including Clostridium perfringens, Clostridium botulinum, and Bacillus spp., which suggests the potential application of the holin gene to study prophage carriage in other bacteria. This study reveals the high incidence of prophage carriage in clinically relevant strains of C. difficile and correlates the molecular data to the morphological observation.     

Integration site for Streptomyces phage phiBT1 and development of site-specific integrating vectors

Despite extensive similarities between the genomes of the Streptomyces temperate phages phiC31 and phiBT1, the attP-int loci are poorly conserved. Here we demonstrate that phiBT1 integrates into a different attachment site than phiC31. phiBT1 attB lies within SCO4848 encoding a 79-amino-acid putative integral membrane protein. Integration vectors based on phiBT1 integrase were shown to have a broad host range and are fully compatible with those based on the phiC31 attP-int locus.     

Genomic organization and molecular characterization of Clostridium difficile bacteriophage PhiCD119

In this study, we have isolated a temperate phage (PhiCD119) from a pathogenic Clostridium difficile strain and sequenced and annotated its genome. This virus has an icosahedral capsid and a contractile tail covered by a sheath and contains a double-stranded DNA genome. It belongs to the Myoviridae family of the tailed phages and the order Caudovirales. The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. The DNA sequence of this phage consists of 53,325 bp, which carries 79 putative open reading frames (ORFs). A function could be assigned to 23 putative gene products, based upon bioinformatic analyses. The PhiCD119 genome is organized in a modular format, which includes modules for lysogeny, DNA replication, DNA packaging, structural proteins, and host cell lysis. The PhiCD119 attachment site attP lies in a noncoding region close to the putative integrase (int) gene. We have identified the phage integration site on the C. difficile chromosome (attB) located in a noncoding region just upstream of gene gltP, which encodes a carrier protein for glutamate and aspartate. This genetic analysis represents the first complete DNA sequence and annotation of a C. difficile phage.     

Physical mapping of Streptomyces coelicolar A3(3) actinophages. III. Restriction analysis

It has been shown that endonucleases HindII, HindIII, SalI and BsuI treatment of phiC62, or phiC43 and phiC31 DNAs forms more than 20 fragments. EcoRI cleaves phiC62, phiC31, phiC31c5 and phiC31c28 into seven fragments, but phiC311yg33 into six fragments. Comparison of molecular weights of DNA restricts obtained after hydrolysis of phage DNAs containing deletions by endonuclease EcoRI made it possible to determine the location of four fragments on restriction map and to orientate this map in relation to the molecule's ends. BamHI cleaves phiC43 DNA into two fragments. By heteroduplexing BamHi site was mapped within the phiC43 insertion sequence.     

In vivo lysogenization of a Clostridium difficile bacteriophage ФCD119

Clostridium difficile is a nosocomial pathogen identified as the cause of antibiotic-associated diarrhea and colitis. In this study, we have documented the lysogeny of a C.?difficile bacteriophage in hamsters during C.?difficile infection. The lysogens isolated from the hamsters were toxin typed and their phage integration site was confirmed by PCR. Through toxin ELISA it was found that the toxin production in the in?vivo isolated lysogens was affected due to ФCD119 lysogenization as in the case of in?vitro isolated ФCD119 lysogens. Together our findings indicate that a baceriophage can lysogenize its C.?difficile host even during the infection process and highlights the importance of lysogeny of C.?difficile phages as an evolutionary adaptation for survival.     

Inactivation of Nocardiophages φC and φEC by Extracts of Bacteriophage-Attachable Cells

Cultures of several species of Nocardia, including N. erythropolis Mat-Ce and Mat-cE mating strains, were extracted with solvents in an attempt to isolate an inactivating complex for nocardiophages phiC and phiEC. Ethanol was the only solvent found effective in solubilizing an inhibitory substance. Inactivating extracts were obtained from the cells of all species to which the phage were able to attach. After extraction of whole cells or cell wall preparations, the phage could not effectively attach to them. Both phages phiC and phiEC were inactivated by the same complex. However, phage phiEC inactivation was 10-fold greater than phiC inactivation. The velocity of inactivation was about 4.1 x 10(2) plaque-forming units per microgram per minute for phiC and 1.1 x 10(3) plaque-forming units per microgram per minute for phage phiEC. The cell extracts required divalent cations for phage inactivation. The inhibitory capacity of the cell extracts was reduced or lost by the activity of proteolytic enzymes, Tween 80, 2-mercaptoethanol, thymol, and sodium lauryl sulfate. Boiling the extract for 10 min did not alter its activity. The inactivating substance was postulated to be a lipoprotein of considerable complexity, unique in the ease with which it is solubilized from host cells by ethanol.     

Mutational Analysis of Highly Conserved Residues in the Phage PhiC31 Integrase Reveals Key Amino Acids Necessary for the DNA Recombination

Background

Amino acid sequence alignment of phage phiC31 integrase with the serine recombinases family revealed highly conserved regions outside the catalytic domain. Until now, no system mutational or biochemical studies have been carried out to assess the roles of these conserved residues in the recombinaton of phiC31 integrase.

Methodology/Principal Findings

To determine the functional roles of these conserved residues, a series of conserved residues were targeted by site-directed mutagenesis. Out of the 17 mutants, 11 mutants showed impaired or no recombination ability, as analyzed by recombination assay both in vivo and in vitro. Results of DNA binding activity assays showed that mutants (R18A, I141A, L143A,E153A, I432A and V571A) exhibited a great decrease in DNA binding affinity, and mutants (G182A/F183A, C374A, C376A/G377A, Y393A and V566A) had completely lost their ability to bind to the specific target DNA attB as compared with wild-type protein. Further analysis of mutants (R18A, I141A, L143A and E153A) synapse and cleavage showed that these mutants were blocked in recombination at the stage of strand cleavage.

Conclusions/Significance

This data reveals that some of the highly conserved residues both in the N-terminus and C-terminus region of phiC31 integrase, play vital roles in the substrate binding and cleavage. The cysteine-rich motif and the C-tail val-rich region of phiC31 integrase may represent the major DNA binding domains of phiC31 integrase.     

Synthesis of bacteriophage phiC DNA in dna mutants of Esherichia coli.

Host dna functions involved in the replication of microvirid phage phiC DNA were investigated in vivo. Although growth of this phage was markedly inhibited even at 35-37 degrees C even in dna+ host, conversion of the infecting single-stranded DNA into the double-stranded parental replicative form (stage I synthesis) occurred normally at 43 degrees C in dna+, dnaA, dnaB, dnaC(D), and dnaE cells. In dnaG mutant, the stage I synthesis was severely inhibited at 43 degrees C but not at 30 degrees C. The stage I replication of phiC DNA was clearly thermosensitive in dnaZ cells incubated in nutrient broth. In Tris-casamino acids-glucose medium, however, the dnaZ mutant sufficiently supported synthesis of the parental replicative form. At 43 degrees C, synthesis of the progeny replicative form DNA (stage II replication) was significantly inhibited even in dna+ cells and was nearly completely blocked in dnaB or dnaC(D) mutant. At 37 degrees C, the stage II replication proceeded normally in dna+ bacteria.     

Transgenic Xenopus laevis embryos can be generated using phiC31 integrase

Bacteriophage phiC31 encodes an integrase that can mediate the insertion of extrachromosomal DNA into genomic DNA. Here we show that the coinjection of mRNA encoding phiC31 integrase with plasmid DNA encoding the green fluorescent protein (GFP) can be used to generate transgenic X. laevis embryos. Despite integration into the genome, appropriate promoter expression required modification of the reporter plasmid by bracketing the GFP reporter gene with tandem copies of the chicken beta-globin 5' HS4 insulator to relieve silencing owing to chromatin position effects. These experiments demonstrate that the integration of insulated gene sequences using phiC31 integrase can be used to efficiently create transgenic embryos in X. laevis and may increase the practical use of phiC31 integrase in other systems as well.     

Targeted engineering of the Drosophila genome

The application of phiC31 phage integrase in Drosophila for unidirectional and site-specific DNA integration was pioneered by Groth et al. in 2004 1 and quickly triggered a wave of innovative tools taking advantage of these unique properties of phiC31. Three recent papers have further developed novel approaches that combine the phiC31-mediated DNA integration with the homologous recombination (HR)-based gene targeting 2 3 for the purpose of efficient and targeted modifications of Drosophila genomic loci. Despite significant differences, the general strategies are similar in principle in the SIRT (site-specific integrase mediated repeated targeting) approach by Gao et al. 4, the IMAGO (integrase-mediated approach for gene knock-out) approach by Choi et al. 5 and the genomic engineering approach developed by our group 6. All three use HR-based gene targeting to first implant a single or a pair of phiC31-attP recombination sites into the target locus. Flies carrying such targeted insertions of attP sites can then be used as "founder lines", in which modified DNA sequences ("knock-in DNA") can be repeatedly and efficiently inserted back into the target locus via phiC31-mediated integration. Thus, by carrying out the targeting experiments only once, one can then directedly and efficiently modify the target locus into virtually any desired knock-in allele. Here we give a brief overview of the SIRT, IMAGO, and genomic engineering approaches and propose a revised genomic engineering scheme in which a single ends-out targeting event will generate founder lines suitable for both recombinase-mediated cassette exchange (RMCE) and single-site based integration of knock-in DNA.     

Genomic fingerprinting of Clostridium difficile isolates by using a random amplified polymorphic DNA (RAPD) assay

Abstract This study describes the use of a new and easy method called random amplfied polymorphic DNA (RAPD) assay to distinguish strains of C. difficile . We used two single short primers (AP4 and AP5) with arbitrary nucleotide sequences in a polymerase chain reaction to amplify genomic DNA. The profiles observed after electrophoretic separation were able to distinguish 20 reference C. difficile strains previously serotyped by Delmées method. The fingerprints of 11 epidemiologically unrelated C. diffiile strains clearly yielded a DNA polymorphism between all the strains. Latterly, RAPD profiles of 11 C. difficile strains isolated from 2 independant suspected outbreaks showed, in each case, a predominant banding pattern correponding to an epidemic strain. These results suggest that RAPD assay could be a valuable tool for epidemiological studies.     

Identification and molecular cloning of a 70 kDa species-specific antigen common to Clostridium difficile

Abstract Three common antigens (CB 1, 2 and 3), characteristic of Clostridium difficile species were identified by immunoblot analysis using homologous and heterologous rabbit antisera, raised against whole cells from 9 distinct strains of C. difficile . A gene library of C. difficile genomic DNA was constructed in Escherichia coli by cloning in Sau 3A-cleaved clostridial DNA fragments into the bacteriophage vector λEMBL3. Out of 3000 plaques screened using the whole cell antisera, 27 clones were positively identified. One of these clones, designated λCd21, expressed high levels of an antigen which could be immunologically identified using whole cell antisera against the 9 C. difficile strains. Antiserum raised against the clone λCd21 identified a 70 kDa antigen (previously named CB1) as demonstrated by immunoblot analysis. Monospecific antiserum against λCd21 recognises the 70 kDa antigen in all 97 strains of C. difficile derived from worldwide sources and does not cross-react with 17 strains from 13 other clostridial species.     

Comparative phage genomics and the evolution of Siphoviridae: insights from dairy phages

Comparative phage genomics can retrace part of the evolutionary history of phage modules encoding phage-specific functions such as capsid building or establishment of the lysogenic state. The diagnosis of relatedness is not based exclusively on sequence similarity, but includes topological considerations of genome organization. The gene maps from the lambda-, psiM2-, L5-, Sfi21-, Sfi11-, phiC31-, sk1- and TM4-like phages showed a remarkable synteny of their structural genes defining a lambda supergroup within Siphoviridae (Caudovirales with long non-contractile tails). A hierarchy of relatedness within the lambda supergroup suggested elements of vertical evolution in the capsid module of Siphoviridae. Links to P22-like Podoviridae and P2-like Myoviridae were also detected. Numerous cases of horizontal gene transfer were observed, but recent transfers were limited to interbreeding phage populations. We suggest that tailed phages are the result of both vertical and horizontal evolution and are thus a good model system for web-like phylogenies.     

Characterization of Temperate Actinophage φC31 Isolated from Streptomyces coelicolor A3(2)

Actinophage phiC31 isolated from Streptomyces coelicolor A3(2), the only strain among actinomycetes for which a genetic map had been constructed, appears to be a typical temperate phage. After phiC31 infection, true lysogenic cultures arose which liberated phage and were immune to infection with homologous phage after repeated single-colony isolations and treatment with phage-specific antiserum. Clear-plaque (c) mutants were derived from phiC31 phage which failed to lysogenize sensitive cultures. Actinophage phiC31 has a temperature-sensitive stage of reproduction. A phage which reproduces with the same effectiveness at high (37 C) and low (28 C) temperatures has also been obtained. Heat-inducible (ct) mutants were isolated from this phage which were able to lysogenize sensitive cultures at 28 C but failed to do so at 37 C. Properties of ct mutants suggest that ct mutations involve a gene controlling maintenance of the lysogenic state in actinomycetes and synthesizing repressor, which may become heat-sensitive as a result of mutation.     

Identification and molecular cloning of a 70 kDa species-specific antigen common to Clostridium difficile

Three common antigens (CB 1, 2 and 3), characteristic of Clostridium difficile species were identified by immunoblot analysis using homologous and heterologous rabbit antisera, raised against whole cells from 9 distinct strains of C. difficile. A gene library of C. difficile genomic DNA was constructed in Escherichia coli by cloning in Sau 3A-cleaved clostridial DNA fragments into the bacteriophage vector lambda EMBL3. OUt of 3000 plaques screened using the whole cell antisera, 27 clones were positively identified. One of these clones, designated gamma Cd21, expressed high levels of an antigen which could be immunologically identified using whole cell antisera against the 9 C. difficile strains. Antiserum raised against the clone gamma Cd21 identified a 70 kDa antigen (previously named CB1) as demonstrated by immunoblot analysis. Monospecific antiserum against gamma Cd21 recognises the 70 kDa antigen in all 97 strains of C. difficile derived from worldwide sources and does not cross-react with 17 strains from 13 other clostridial species.     

Transgene excision from wheat chromosomes by phage phiC31 integrase

The Streptomyces phage phiC31 integrase was tested for its ability to excise transgenic DNA from the wheat genome by site-specific recombination. Plants that stably express phiC31 integrase were crossed to plants carrying a target construct bearing the phiC31 recognition sites, attP and attB. In the progeny, phiC31 recombinase mediates recombination between the att sites of the target locus, which results in excision of the intervening DNA. Recombination events could be identified in 34 independent wheat lines by PCR and Southern blot analysis and by sequencing of the excision footprints. Recombinant loci were inherited to the subsequent generation. The results presented here establish the integrase-att system as a tool for catalysing the precise elimination of DNA sequences from wheat chromosomes.