Search tips
Search criteria

Results 1-25 (581425)

Clipboard (0)

Related Articles

1.  Genomic Analysis of Clostridium perfringens Bacteriophage φ3626, Which Integrates into guaA and Possibly Affects Sporulation 
Journal of Bacteriology  2002;184(16):4359-4368.
Two temperate viruses, φ3626 and φ8533, have been isolated from lysogenic Clostridium perfringens strains. Phage φ3626 was chosen for detailed analysis and was inspected by electron microscopy, protein profiling, and host range determination. For the first time, the nucleotide sequence of a bacteriophage infecting Clostridium species was determined. The virus belongs to the Siphoviridae family of the tailed phages, the order Caudovirales. Its genome consists of a linear double-stranded DNA molecule of 33,507 nucleotides, with invariable 3′-protruding cohesive ends of nine residues. Fifty open reading frames were identified, which are organized in three major life cycle-specific gene clusters. The genes required for lytic development show an opposite orientation and arrangement compared to the lysogeny control region. A function could be assigned to 19 gene products, based upon bioinformatic analyses, N-terminal amino acid sequencing, or experimental evidence. These include DNA-packaging proteins, structural components, a dual lysis system, a putative lysogeny switch, and proteins that are involved in replication, recombination, and modification of phage DNA. The presence of genes encoding a putative sigma factor related to sporulation-dependent sigma factors and a putative sporulation-dependent transcription regulator suggests a possible interaction of φ3626 with onset of sporulation in C. perfringens. We found that the φ3626 attachment site attP lies in a noncoding region immediately downstream of int. Integration of the viral genome occurs into the bacterial attachment site attB, which is located within the 3′ end of a guaA homologue. This essential housekeeping gene is functionally independent of the integration status, due to reconstitution of its terminal codons by phage sequence.
PMCID: PMC135250  PMID: 12142405
2.  Evaluating the Involvement of Alternative Sigma Factors SigF and SigG in Clostridium perfringens Sporulation and Enterotoxin Synthesis▿  
Infection and Immunity  2010;78(10):4286-4293.
Clostridium perfringens type A food poisoning is the second most commonly identified bacterial food-borne illness. Sporulation contributes to this disease in two ways: (i) most food-poisoning strains form exceptionally resistant spores to facilitate their survival of food-associated stresses, and (ii) the enterotoxin (CPE) responsible for the symptoms of this food poisoning is synthesized only during sporulation. In Bacillus subtilis, four alternative sigma factors mediate sporulation. The same four sigma factors are encoded by C. perfringens genomes, and two (SigE and SigK) have previously been shown to be necessary for sporulation and CPE production by SM101, a transformable derivative of a C. perfringens food-poisoning strain (K. H. Harry, R. Zhou, L. Kroos, and S. B. Melville, J. Bacteriol. 2009, 191:2728-2742). However, the importance of SigF and SigG for C. perfringens sporulation or CPE production had not yet been assessed. In the current study, after confirming that sporulating wild-type SM101 cultures produce SigF (from a tricistronic operon) and SigG, we prepared isogenic sigF- or sigG-null mutants. Whereas SM101 formed heat-resistant, phase-refractile spores, spore formation was blocked in the sigF- and sigG-null mutants. Complementation fully restored sporulation by both mutants. By use of these mutants and complementing strains, CPE production was shown to be SigF dependent but SigG independent. This finding apparently involved regulation of the production of SigE and SigK, which Harry et al. showed to be necessary for CPE synthesis, by SigF. By combining these findings with those previous results, it is now apparent that all four alternative sigma factors are necessary for C. perfringens sporulation, but only SigE, SigF, and SigK are needed for CPE synthesis.
PMCID: PMC2950359  PMID: 20643850
3.  Involvement of Clostridium botulinum ATCC 3502 Sigma Factor K in Early-Stage Sporulation 
Applied and Environmental Microbiology  2012;78(13):4590-4596.
A key survival mechanism of Clostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organism Bacillus subtilis, nothing is known about these mechanisms in C. botulinum. Using the ClosTron gene-knockout tool, sigK, encoding late-stage (stage IV) sporulation sigma factor K in B. subtilis, was disrupted in C. botulinum ATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast, sigK-complemented mutants sporulated successfully. Quantitative real-time PCR analysis of sigK in the parent strain revealed expression at the late log growth phase in the parent strain. Analysis of spo0A, encoding the sporulation master switch, in the sigK mutant and the parent showed significantly reduced relative levels of spo0A expression in the sigK mutant compared to the parent strain. Similarly, sigF showed significantly lower relative transcription levels in the sigK mutant than the parent strain, suggesting that the sporulation pathway was blocked in the sigK mutant at an early stage. We conclude that σK is essential for early-stage sporulation in C. botulinum ATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organism B. subtilis. Understanding the sporulation mechanism of C. botulinum provides keys to control the public health risks that the spores of this dangerous pathogen cause through foods.
PMCID: PMC3370484  PMID: 22544236
4.  Identification and Characterization of Sporulation-Dependent Promoters Upstream of the Enterotoxin Gene (cpe) of Clostridium perfringens 
Journal of Bacteriology  1998;180(1):136-142.
Three promoter sites (P1, P2, and P3) responsible for the sporulation-associated synthesis of Clostridium perfringens enterotoxin, a common cause of food poisoning in humans and animals, were identified. Nested and internal deletions of the cpe promoter region were made to narrow down the location of promoter elements. To measure the effects of the deletions on the expression of cpe, translational fusions containing the promoter deletions were made with the gusA gene of Escherichia coli, which codes for β-glucuronidase; E. coli-C. perfringens shuttle vectors carrying the fusions were introduced into C. perfringens by electroporation. In addition, in vitro transcription assays were performed with the cpe promoter region as the DNA template for extracts made from sporulating cells. DNA sequences upstream of P1 were similar to consensus SigK-dependent promoters, while P2 and P3 were similar to consensus SigE-dependent promoters. SigE and SigK are sporulation-associated sigma factors known to be active in the mother cell compartment of sporulating cells of Bacillus subtilis, the same compartment in which enterotoxin is synthesized in C. perfringens.
PMCID: PMC106859  PMID: 9422603
5.  Sporulation and Enterotoxin (CPE) Synthesis Are Controlled by the Sporulation-Specific Sigma Factors SigE and SigK in Clostridium perfringens▿ ‡  
Journal of Bacteriology  2009;191(8):2728-2742.
Clostridium perfringens is the third most frequent cause of bacterial food poisoning annually in the United States. Ingested C. perfringens vegetative cells sporulate in the intestinal tract and produce an enterotoxin (CPE) that is responsible for the symptoms of acute food poisoning. Studies of Bacillus subtilis have shown that gene expression during sporulation is compartmentalized, with different genes expressed in the mother cell and the forespore. The cell-specific RNA polymerase sigma factors σF, σE, σG, and σK coordinate much of the developmental process. The C. perfringens cpe gene, encoding CPE, is transcribed from three promoters, where P1 was proposed to be σK dependent, while P2 and P3 were proposed to be σE dependent based on consensus promoter recognition sequences. In this study, mutations were introduced into the sigE and sigK genes of C. perfringens. With the sigE and sigK mutants, gusA fusion assays indicated that there was no expression of cpe in either mutant. Results from gusA fusion assays and immunoblotting experiments indicate that σE-associated RNA polymerase and σK-associated RNA polymerase coregulate each other's expression. Transcription and translation of the spoIIID gene in C. perfringens were not affected by mutations in sigE and sigK, which differs from B. subtilis, in which spoIIID transcription requires σE-associated RNA polymerase. The results presented here show that the regulation of developmental events in the mother cell compartment of C. perfringens is not the same as that in B. subtilis and Clostridium acetobutylicum.
PMCID: PMC2668419  PMID: 19201796
6.  Site-Specific Recombination of Temperate Myxococcus xanthus Phage Mx8: Regulation of Integrase Activity by Reversible, Covalent Modification† 
Journal of Bacteriology  1999;181(13):4062-4070.
Temperate Myxococcus xanthus phage Mx8 integrates into the attB locus of the M. xanthus genome. The phage attachment site, attP, is required in cis for integration and lies within the int (integrase) coding sequence. Site-specific integration of Mx8 alters the 3′ end of int to generate the modified intX gene, which encodes a less active form of integrase with a different C terminus. The phage-encoded (Int) form of integrase promotes attP × attB recombination more efficiently than attR × attB, attL × attB, or attB × attB recombination. The attP and attB sites share a common core. Sequences flanking both sides of the attP core within the int gene are necessary for attP function. This information shows that the directionality of the integration reaction depends on arm sequences flanking both sides of the attP core. Expression of the uoi gene immediately upstream of int inhibits integrative (attP × attB) recombination, supporting the idea that uoi encodes the Mx8 excisionase. Integrase catalyzes a reaction that alters the primary sequence of its gene; the change in the primary amino acid sequence of Mx8 integrase resulting from the reaction that it catalyzes is a novel mechanism by which the reversible, covalent modification of an enzyme is used to regulate its specific activity. The lower specific activity of the prophage-encoded IntX integrase acts to limit excisive site-specific recombination in lysogens carrying a single Mx8 prophage, which are less immune to superinfection than lysogens carrying multiple, tandem prophages. Thus, this mechanism serves to regulate Mx8 site-specific recombination and superinfection immunity coordinately and thereby to preserve the integrity of the lysogenic state.
PMCID: PMC93897  PMID: 10383975
7.  Unique Regulatory Mechanism of Sporulation and Enterotoxin Production in Clostridium perfringens 
Journal of Bacteriology  2013;195(12):2931-2936.
Clostridium perfringens causes gas gangrene and gastrointestinal (GI) diseases in humans. The most common cause of C. perfringens-associated food poisoning is the consumption of C. perfringens vegetative cells followed by sporulation and production of enterotoxin in the gut. Despite the importance of spore formation in C. perfringens pathogenesis, the details of the regulation of sporulation have not yet been defined fully. In this study, microarray and bioinformatic analyses identified a candidate gene (the RNA regulator virX) for the repression of genes encoding positive regulators (Spo0A and sigma factors) of C. perfringens sporulation. A virX mutant constructed in the food poisoning strain SM101 had a much higher sporulation efficiency than that of the wild type. The transcription of sigE, sigF, and sigK was strongly induced at 2.5 h of culture of the virX mutant. Moreover, the transcription of the enterotoxin gene was also strongly induced in the virX mutant. Western blotting confirmed that the levels of enterotoxin production were higher in the virX mutant than in the wild type. These observations indicated that the higher levels of sporulation and enterotoxin production in the virX mutant were specifically due to inactivation of the virX gene. Since virX homologues were not found in any Bacillus species but were present in other clostridial species, our findings identify further differences in the regulation of sporulation between Bacillus and certain Clostridium species. The virX RNA regulator plays a key role in the drastic shift in lifestyle of the anaerobic flesh eater C. perfringens between the vegetative state (for gas gangrene) and the sporulating state (for food poisoning).
PMCID: PMC3697249  PMID: 23585540
8.  Site-Specific Recombination of Temperate Myxococcus xanthus Phage Mx8: Genetic Elements Required for Integration† 
Journal of Bacteriology  1999;181(13):4050-4061.
Like most temperate bacteriophages, phage Mx8 integrates into a preferred locus on the genome of its host, Myxococcus xanthus, by a mechanism of site-specific recombination. The Mx8 int-attP genes required for integration map within a 2.2-kilobase-pair (kb) fragment of the phage genome. When this fragment is subcloned into a plasmid vector, it facilitates the site-specific integration of the plasmid into the 3′ ends of either of two tandem tRNAAsp genes, trnD1 and trnD2, located within the attB locus of the M. xanthus genome. Although Int-mediated site-specific recombination occurs between attP and either attB1 (within trnD1) or attB2 (within trnD2), the attP × attB1 reaction is highly favored and often is accompanied by a deletion between attB1 and attB2. The int gene is the only Mx8 gene required in trans for attP × attB recombination. The int promoter lies within the 106-bp region immediately upstream of one of two alternate GTG start codons, GTG-5208 (GTG at bp 5208) and GTG-5085, for integrase and likely is repressed in the prophage state. All but the C-terminal 30 amino acid residues of the Int protein are required for its ability to mediate attP × attB recombination efficiently. The attP core lies within the int coding sequence, and the product of integration is a prophage in which the 3′ end of int is replaced by host sequences. The prophage intX gene is predicted to encode an integrase with a different C terminus.
PMCID: PMC93896  PMID: 10383974
9.  Isolation of New Stenotrophomonas Bacteriophages and Genomic Characterization of Temperate Phage S1▿  
Applied and Environmental Microbiology  2008;74(24):7552-7560.
Twenty-two phages that infect Stenotrophomonas species were isolated through sewage enrichment and prophage induction. Of them, S1, S3, and S4 were selected due to their wide host ranges compared to those of the other phages. S1 and S4 are temperate siphoviruses, while S3 is a virulent myovirus. The genomes of S3 and S4, about 33 and 200 kb, were resistant to restriction digestion. The lytic cycles lasted 30 min for S3 and about 75 min for S1 and S4. The burst size for S3 was 100 virions/cell, while S1 and S4 produced about 75 virus particles/cell. The frequency of bacteriophage-insensitive host mutants, calculated by dividing the number of surviving colonies by the bacterial titer of a parallel, uninfected culture, ranged between 10−5 and 10−6 for S3 and 10−3 and 10−4 for S1 and S4. The 40,287-bp genome of S1 contains 48 open reading frames (ORFs) and 12-bp 5′ protruding cohesive ends. By using a combination of bioinformatics and experimental evidence, functions were ascribed to 21 ORFs. The morphogenetic and lysis modules are well-conserved, but no lysis-lysogeny switch or DNA replication gene clusters were recognized. Two major clusters of genes with respect to transcriptional orientation were observed. Interspersed among them were lysogenic conversion genes encoding phosphoadenosine phosphosulfate reductase and GspM, a protein involved in the general secretion system II. The attP site of S1 may be located within a gene that presents over 75% homology to a Stenotrophomonas chromosomal determinant.
PMCID: PMC2607143  PMID: 18952876
10.  Tight Regulation of the intS Gene of the KplE1 Prophage: A New Paradigm for Integrase Gene Regulation 
PLoS Genetics  2010;6(10):e1001149.
Temperate phages have the ability to maintain their genome in their host, a process called lysogeny. For most, passive replication of the phage genome relies on integration into the host's chromosome and becoming a prophage. Prophages remain silent in the absence of stress and replicate passively within their host genome. However, when stressful conditions occur, a prophage excises itself and resumes the viral cycle. Integration and excision of phage genomes are mediated by regulated site-specific recombination catalyzed by tyrosine and serine recombinases. In the KplE1 prophage, site-specific recombination is mediated by the IntS integrase and the TorI recombination directionality factor (RDF). We previously described a sub-family of temperate phages that is characterized by an unusual organization of the recombination module. Consequently, the attL recombination region overlaps with the integrase promoter, and the integrase and RDF genes do not share a common activated promoter upon lytic induction as in the lambda prophage. In this study, we show that the intS gene is tightly regulated by its own product as well as by the TorI RDF protein. In silico analysis revealed that overlap of the attL region with the integrase promoter is widely encountered in prophages present in prokaryotic genomes, suggesting a general occurrence of negatively autoregulated integrase genes. The prediction that these integrase genes are negatively autoregulated was biologically assessed by studying the regulation of several integrase genes from two different Escherichia coli strains. Our results suggest that the majority of tRNA-associated integrase genes in prokaryotic genomes could be autoregulated and that this might be correlated with the recombination efficiency as in KplE1. The consequences of this unprecedented regulation for excisive recombination are discussed.
Author Summary
Temperate bacteriophages are widespread bacterial viruses that have the ability to replicate passively in their hosts as long as no stressful conditions are encountered, a process called lysogeny. Prophage-encoded genes may benefit the host in several ways such as providing resistance to antibiotics, increased pathogenicity, or increased fitness. Most temperate phages insert their genome into the host's chromosome by site-specific recombination. After prophage induction, usually under stressful conditions, the excisive recombination constitutes a key step toward productive phage development. In this paper, we study the regulation of integrase genes that encode the enzyme required for integrative as well as excisive recombination. We noticed that for prophages inserted in or near tRNA genes the orientation of the integrase gene relative to the tRNA is crucial for its regulation.
PMCID: PMC2951348  PMID: 20949106
11.  Genomic Organization and Molecular Characterization of Clostridium difficile Bacteriophage ΦCD119 
Journal of Bacteriology  2006;188(7):2568-2577.
In this study, we have isolated a temperate phage (ΦCD119) 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 ΦCD119 genome is organized in a modular format, which includes modules for lysogeny, DNA replication, DNA packaging, structural proteins, and host cell lysis. The ΦCD119 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.
PMCID: PMC1428422  PMID: 16547044
12.  Developmentally-Regulated Excision of the SPβ Prophage Reconstitutes a Gene Required for Spore Envelope Maturation in Bacillus subtilis 
PLoS Genetics  2014;10(10):e1004636.
Temperate phages infect bacteria by injecting their DNA into bacterial cells, where it becomes incorporated into the host genome as a prophage. In the genome of Bacillus subtilis 168, an active prophage, SPβ, is inserted into a polysaccharide synthesis gene, spsM. Here, we show that a rearrangement occurs during sporulation to reconstitute a functional composite spsM gene by precise excision of SPβ from the chromosome. SPβ excision requires a putative site-specific recombinase, SprA, and an accessory protein, SprB. A minimized SPβ, where all the SPβ genes were deleted, except sprA and sprB, retained the SPβ excision activity during sporulation, demonstrating that sprA and sprB are necessary and sufficient for the excision. While expression of sprA was observed during vegetative growth, sprB was induced during sporulation and upon mitomycin C treatment, which triggers the phage lytic cycle. We also demonstrated that overexpression of sprB (but not of sprA) resulted in SPβ prophage excision without triggering the lytic cycle. These results suggest that sprB is the factor that controls the timing of phage excision. Furthermore, we provide evidence that spsM is essential for the addition of polysaccharides to the spore envelope. The presence of polysaccharides on the spore surface renders the spore hydrophilic in water. This property may be beneficial in allowing spores to disperse in natural environments via water flow. A similar rearrangement occurs in Bacillus amyloliquefaciens FZB42, where a SPβ-like element is excised during sporulation to reconstitute a polysaccharide synthesis gene, suggesting that this type of gene rearrangement is common in spore-forming bacteria because it can be spread by phage infection.
Author Summary
Integration of prophages into protein-coding sequences of the host chromosome generally results in loss of function of the interrupted gene. In the endospore-forming organism Bacillus subtilis strain 168, the SPβ prophage is inserted into a previously-uncharacterized spore polysaccharide synthesis gene, spsM. In vegetative cells, the lytic cycle is induced in response to DNA damage. In the process, SPβ is excised from the genome to form phage particles. Here, we demonstrate that SPβ excision is also a developmentally-regulated event that occurs systematically during sporulation to reconstitute a functional spsM gene. Following asymmetric division of the sporulating cell, two cellular compartments are generated, the forespore, which will mature into a spore, and the mother cell, which is essential to the process of spore maturation. Because phage excision is limited to the mother cell genome, and does not occur in the forespore genome, SPβ is an integral part of the spore genome. Thus, after the spores germinate, the vegetative cells resume growth and the SPβ prophage is propagated vertically to the progeny along with the rest of the host genome. Our results suggest that the two pathways of SPβ excision support both the phage life cycle and normal sporulation of the host cells.
PMCID: PMC4191935  PMID: 25299644
13.  Lysogeny and Sporulation in Bacillus Isolates from the Gulf of Mexico▿ †  
Eleven Bacillus isolates from the surface and subsurface waters of the Gulf of Mexico were examined for their capacity to sporulate and harbor prophages. Occurrence of sporulation in each isolate was assessed through decoyinine induction, and putative lysogens were identified by prophage induction by mitomycin C treatment. No obvious correlation between ability to sporulate and prophage induction was found. Four strains that contained inducible virus-like particles (VLPs) were shown to sporulate. Four strains did not produce spores upon induction by decoyinine but contained inducible VLPs. Two of the strains did not produce virus-like particles or sporulate significantly upon induction. Isolate B14905 had a high level of virus-like particle production and a high occurrence of sporulation and was further examined by genomic sequencing in an attempt to shed light on the relationship between sporulation and lysogeny. In silico analysis of the B14905 genome revealed four prophage-like regions, one of which was independently sequenced from a mitomycin C-induced lysate. Based on PCR and transmission electron microscopy (TEM) analysis of an induced phage lysate, one is a noninducible phage remnant, one may be a defective phage-like bacteriocin, and two were inducible prophages. One of the inducible phages contained four putative transcriptional regulators, one of which was a SinR-like regulator that may be involved in the regulation of host sporulation. Isolates that both possess the capacity to sporulate and contain temperate phage may be well adapted for survival in the oligotrophic ocean.
PMCID: PMC2812986  PMID: 20008174
14.  Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae 
PLoS ONE  2012;7(5):e38283.
Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae.
PMCID: PMC3362512  PMID: 22666499
15.  Brochothrix thermosphacta Bacteriophages Feature Heterogeneous and Highly Mosaic Genomes and Utilize Unique Prophage Insertion Sites ▿ †  
Journal of Bacteriology  2010;192(20):5441-5453.
Brochothrix belongs to the low-GC branch of Gram-positive bacteria (Firmicutes), closely related to Listeria, Staphylococcus, Clostridium, and Bacillus. Brochothrix thermosphacta is a nonproteolytic food spoilage organism, adapted to growth in vacuum-packaged meats. We report the first genome sequences and characterization of Brochothrix bacteriophages. Phage A9 is a myovirus with an 89-nm capsid diameter and a 171-nm contractile tail; it belongs to the Spounavirinae subfamily and shares significant homologies with Listeria phage A511, Staphylococcus phage Twort, and others. The A9 unit genome is 127 kb long with 11-kb terminal redundancy; it encodes 198 proteins and 6 tRNAs. Phages BL3 and NF5 are temperate siphoviruses with a head diameter of 56 to 59 nm. The BL3 tail is 270 nm long, whereas NF5 features a short tail of only 94 nm. The NF5 genome (36.95 kb) encodes 57 gene products, BL3 (41.52 kb) encodes 65 products, and both are arranged in life cycle-specific modules. Surprisingly, BL3 and NF5 show little relatedness to Listeria phages but rather demonstrate relatedness to lactococcal phages. Peptide mass fingerprinting of viral proteins indicate programmed −1 translational frameshifts in the NF5 capsid and the BL3 major tail protein. Both NF5 and BL3 feature circularly permuted, terminally redundant genomes, packaged by a headful mechanism, and integrases of the serine (BL3) and tyrosine (NF5) types. They utilize unique target sequences not previously described: BL3 inserts into the 3′ end of a RNA methyltransferase, whereas NF5 integrates into the 5′-terminal part of a putative histidinol-phosphatase. Interestingly, both genes are reconstituted by phage sequence.
PMCID: PMC2950505  PMID: 20709901
16.  Construction of an Integration-Proficient Vector Based on the Site-Specific Recombination Mechanism of Enterococcal Temperate Phage φFC1 
Journal of Bacteriology  2002;184(7):1859-1864.
The genome of temperate phage φFC1 integrates into the chromosome of Enterococcus faecalis KBL 703 via site-specific recombination. In this study, an integration vector containing the attP site and putative integrase gene mj1 of phage φFC1 was constructed. A 2,744-bp fragment which included the attP site and mj1 was inserted into a pUC19 derivative containing the cat gene to construct pEMJ1-1. E. faecalis KBL 707, which does not contain the bacteriophage but which has a putative attB site within its genome, could be transformed by pEMJ1-1. Southern hybridization, PCR amplification, and DNA sequencing revealed that pEMJ1-1 was integrated specifically at the putative attB site within the E. faecalis KBL 707 chromosome. This observation suggested that the 2,744-bp fragment carrying mj1 and the attP site of phage φFC1 was sufficient for site-specific recombination and that pEMJ1-1 could be used as a site-specific integration vector. The transformation efficiency of pEMJ1-1 was as high as 6 × 103 transformants/μg of DNA. In addition, a vector (pATTB1) containing the 290-bp attB region was constructed. pATTB1 was transformed into Escherichia coli containing a derivative of the pET14b vector carrying attP and mj1. This resulted in the formation of chimeric plasmids by site-specific recombination between the cloned attB and attP sequences. The results indicate that the integration vector system based on the site-specific recombination mechanism of phage φFC1 can be used for genetic engineering in E. faecalis and in other hosts.
PMCID: PMC134912  PMID: 11889091
17.  Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium1 
Poultry science  2013;92(2):526-533.
There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently used antibiotics. Clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium that plays a significant role in human foodborne disease as well as nonfoodborne human, animal, and avian diseases. Countries that have complied with the ban on antimicrobial growth promoters in feeds have reported increased incidences of C. perfringens-associated diseases in poultry. To address these issues, new antimicrobial agents, putative lysins encoded by the genomes of bacteriophages, are being identified in our laboratory. Poultry intestinal material, soil, sewage, and poultry processing drainage water were screened for virulent bacteriophages that could lyse C. perfringens and produce clear plaques in spot assays. Bacteriophages were isolated that had long noncontractile tails, members of the family Siphoviridae, and with short noncontractile tails, members of the family Podoviridae. Several bacteriophage genes were identified that encoded N-acetylmuramoyl-l-alanine amidases, lysozyme-endopeptidases, and a zinc carboxypeptidase domain that has not been previously reported in viral genomes. Putative phage lysin genes (ply) were cloned and expressed in Escherichia coli. The recombinant lysins were amidases capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays, but did not lyse any clostridia beyond the species. Consequently, bacteriophage gene products could eventually be used to target bacterial pathogens, such as C. perfringens via a species-specific strategy, to control animal and human diseases without having deleterious effects on beneficial probiotic bacteria.
PMCID: PMC4089029  PMID: 23300321
enzybiotic; antibiotic alternative; bacterial virus; food safety; animal/human health
18.  The Genome of ε15, a Serotype-Converting, Group E1 Salmonella enterica-Specific Bacteriophage 
Virology  2007;369(2):234-244.
The genome sequence of the Salmonella enterica serovar Anatum-specific, serotype-converting bacteriophage ε15 has been completed. The nonredundant genome contains 39,671 bp and 51 putative genes. It most closely resembles the genome of ϕV10, an Escherichia coli O1H57-specific temperate phage, with which it shares 36 related genes. More distant relatives include the Burkholderia cepacia-specific phage, BcepC6B [8 similar genes], the Bordetella bronchiseptica-specific phage, BPP-1 [8 similar genes] and the Photobacterium profundum prophage, P Pϕpr1 [6 similar genes].
ε15 gene identifications based on homologies with known gene families include the terminase small and large subunits, integrase, endolysin, two holins, two DNA methylase enzymes (one adenine-specific and one cytosine-specific) and a RecT-like enzyme. Genes identified experimentally include those coding for the serotype conversion proteins, the tail fiber, the major capsid protein and the major repressor. ε15’s attP site and the Salmonella attB site with which it interacts during lysogenization have also been determined.
PMCID: PMC2698709  PMID: 17825342
bacteriophage; serotype conversion; virion proteins; Epsilon 15 genome; attP; attB
19.  Control of Directionality in Bacteriophage mv4 Site-Specific Recombination: Functional Analysis of the Xis Factor▿  
Journal of Bacteriology  2009;192(3):624-635.
The integrase of the temperate bacteriophage mv4 catalyzes site-specific recombination between the phage attP site and the host attB site during Lactobacillus delbrueckii lysogenization. The mv4 prophage is excised during the induction of lytic growth. Excisive site-specific recombination between the attR and attL sites is also catalyzed by the phage-encoded recombinase, but the directionality of the recombination is determined by a second phage-encoded protein, the recombination directionality factor (RDF). We have identified and functionally characterized the RDF involved in site-specific excision of the prophage genome. The mv4 RDF, mv4Xis, is encoded by the second gene of the early lytic operon. It is a basic protein of 56 amino acids. Electrophoretic mobility shift assays demonstrated that mv4Xis binds specifically to the attP and attR sites via two DNA-binding sites, introducing a bend into the DNA. In vitro experiments and in vivo recombination assays with plasmids in Escherichia coli and Lactobacillus plantarum demonstrated that mv4Xis is absolutely required for inter- or intramolecular recombination between the attR and attL sites. In contrast to the well-known phage site-specific recombination systems, the integrative recombination between the attP and attB sites seems not to be inhibited by the presence of mv4Xis.
PMCID: PMC2812466  PMID: 19948798
20.  Prophage Carriage and Diversity within Clinically Relevant Strains of Clostridium difficile 
Applied and Environmental Microbiology  2012;78(17):6027-6034.
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.
PMCID: PMC3416593  PMID: 22706062
21.  Site-specific integration of the temperate bacteriophage phi adh into the Lactobacillus gasseri chromosome and molecular characterization of the phage (attP) and bacterial (attB) attachment sites. 
Journal of Bacteriology  1992;174(17):5584-5592.
The temperate bacteriophage phi adh integrates its genome into the chromosomal DNA of Lactobacillus gasseri ADH by a site-specific recombination process. Southern hybridization analysis of BclI-digested genomic DNA from six relysogenized derivatives of the prophage-cured strain NCK102 displayed phage-chromosomal junction fragments identical to those of the lysogenic parent. The phi adh attachment site sequence, attP, was located within a 365-bp EcoRI-HindIII fragment of phage phi adh. This fragment was cloned and sequenced. DNA sequence analysis revealed striking features common to the attachment sites of other site-specific recombination systems: five direct repeats of the sequence TGTCCCTTTT(C/T) and a 14-bp inverted repeat. Oligonucleotides derived from the sequence of the attP-containing fragment enabled us to amplify predicted junction fragment sequences and thus to identify attL, attR, and attB. The core region was defined as the 16-bp sequence TACACTTCTTAGGAGG. Phage-encoded functions essential for site-specific insertion of phage phi adh were located in a 4.5-kb BclI fragment. This fragment was cloned in plasmid pSA34 to generate the insertional vector pTRK182. Plasmid pTRK182 was introduced into L. gasseri NCK102 by electroporation. Hybridization analysis showed that a single copy of pTRK182 had integrated at the attB site of the NCK102 erythromycin-resistant transformants. This is the first site-specific recombination system described in lactobacilli, as well as the first attP-based site-specific integration vector constructed for L. gasseri ADH.
PMCID: PMC206502  PMID: 1512192
22.  Novel System for Efficient Isolation of Clostridium Double-Crossover Allelic Exchange Mutants Enabling Markerless Chromosomal Gene Deletions and DNA Integration 
Applied and Environmental Microbiology  2012;78(22):8112-8121.
Isolation of Clostridium mutants based on gene replacement via allelic exchange remains a major limitation for this important genus. Use of a heterologous counterselection marker can facilitate the identification of the generally rare allelic exchange events. We report on the development of an inducible counterselection marker and describe its utility and broad potential in quickly and efficiently generating markerless DNA deletions and integrations at any genomic locus without the need for auxotrophic mutants or the use of the mobile group II introns. This system is based on a codon-optimized mazF toxin gene from Escherichia coli under the control of a lactose-inducible promoter from Clostridium perfringens. This system is potentially applicable to almost all members of the genus Clostridium due to their similarly low genomic GC content and comparable codon usage. We isolated all allelic-exchange-based gene deletions (ca_p0167, sigF, and sigK) or disruptions (ca_p0157 and sigF) we attempted and integrated a 3.6-kb heterologous DNA sequence (made up of a Clostridium ljungdahlii 2.1-kb formate dehydrogenase [fdh] gene plus a FLP recombination target [FRT]-flanked thiamphenicol resistance marker) into the Clostridium acetobutylicum chromosome. Furthermore, we report on the development of a plasmid system with inducible segregational instability, thus enabling efficient deployment of the FLP-FRT system to generate markerless deletion or integration mutants. This enabled expeditious deletion of the thiamphenicol resistance marker from the fdh integrant strain as well as the sigK deletion strain. More generally, our system can potentially be applied to other organisms with underdeveloped genetic tools.
PMCID: PMC3485963  PMID: 22983967
23.  Diverse Temperate Bacteriophage Carriage in Clostridium difficile 027 Strains 
PLoS ONE  2012;7(5):e37263.
The hypervirulent Clostridium difficile ribotype 027 can be classified into subtypes, but it unknown if these differ in terms of severity of C. difficile infection (CDI). Genomic studies of C. difficile 027 strains have established that they are rich in mobile genetic elements including prophages. This study combined physiological studies, electron microscopy analysis and molecular biology to determine the potential role of temperate bacteriophages in disease and diversity of C. difficile 027.
Methodology/Principal Findings
We induced prophages from 91 clinical C. difficile 027 isolates and used transmission electron microscopy and pulsed-field gel electrophoresis to characterise the bacteriophages present. We established a correlation between phage morphology and subtype. Morphologically distinct tailed bacteriophages belonging to Myoviridae and Siphoviridae were identified in 63 and three isolates, respectively. Dual phage carriage was observed in four isolates. In addition, there were inducible phage tail-like particles (PT-LPs) in all isolates. The capacity of two antibiotics mitomycin C and norfloxacin to induce prophages was compared and it was shown that they induced specific prophages from C. difficile isolates. A PCR assay targeting the capsid gene of the myoviruses was designed to examine molecular diversity of C. difficile myoviruses. Phylogenetic analysis of the capsid gene sequences from eight ribotypes showed that all sequences found in the ribotype 027 isolates were identical and distinct from other C. difficile ribotypes and other bacteria species.
A diverse set of temperate bacteriophages are associated with C. difficile 027. The observed correlation between phage carriage and the subtypes suggests that temperate bacteriophages contribute to the diversity of C. difficile 027 and may play a role in severity of disease associated with this ribotype. The capsid gene can be used as a tool to identify C. difficile myoviruses present within bacterial genomes.
PMCID: PMC3356267  PMID: 22624004
24.  Prophage-Stimulated Toxin Production in Clostridium difficile NAP1/027 Lysogens▿† 
Journal of Bacteriology  2011;193(11):2726-2734.
TcdA and TcdB exotoxins are the main virulence factors of Clostridium difficile, one of the most deadly nosocomial pathogens. Recent data suggest that prophages can influence the regulation of toxin expression. Here we present the characterization of φCD38-2, a pac-type temperate Siphoviridae phage that stimulates toxin expression when introduced as a prophage into C. difficile. Host range analysis showed that φCD38-2 was able to infect 99/207 isolates of C. difficile representing 11 different PCR ribotypes. Of 89 isolates corresponding to the NAP1/027 hypervirulent strain, which recently caused several outbreaks in North America and Europe, 79 (89%) were sensitive to φCD38-2. The complete double-stranded DNA (dsDNA) genome was determined, and a putative function could be assigned to 24 of the 55 open reading frames. No toxins or virulence factors could be identified based on bioinformatics analyses. Our data also suggest that φCD38-2 replicates as a circular plasmid in C. difficile lysogens. Upon introduction of φCD38-2 into a NAP1/027 representative isolate, up to 1.6- and 2.1-fold more TcdA and TcdB, respectively, were detected by immunodot blotting in culture supernatants of the lysogen than in the wild-type strain. In addition, real-time quantitative reverse transcriptase PCR (qRT-PCR) analyses showed that the mRNA levels of all five pathogenicity locus (PaLoc) genes were higher in the CD274 lysogen. Our study provides the first genomic sequence of a new pac-type Siphoviridae phage family member infecting C. difficile and brings further evidence supporting the role of prophages in toxin production in this important nosocomial pathogen.
PMCID: PMC3133130  PMID: 21441508
25.  Clostridium perfringens bacteriophages ΦCP39O and ΦCP26F: genomic organization and proteomic analysis of the virions 
Archives of virology  2010;156(1):25-35.
Poultry intestinal material, sewage and poultry processing drainage water were screened for virulent Clostridium perfringens bacteriophages. Viruses isolated from broiler chicken offal washes (O) and poultry feces (F), designated ΦCP39O and ΦCP26F, respectively, produced clear plaques on host strains. Both bacteriophages had isometric heads of 57 nm in diameter with 100-nm non-contractile tails characteristic of members of the family Siphoviridae in the order Caudovirales. The double-strand DNA genome of bacteriophage ΦCP39O was 38,753 base pairs (bp), while the ΦCP26F genome was 39,188 bp, with an average GC content of 30.3%. Both viral genomes contained 62 potential open reading frames (ORFs) predicted to be encoded on one strand. Among the ORFs, 29 predicted proteins had no known similarity while others encoded putative bacteriophage capsid components such as a pre-neck/appendage, tail, tape measure and portal proteins. Other genes encoded a predicted DNA primase, single-strand DNA-binding protein, terminase, thymidylate synthase and a transcription factor. Potential lytic enzymes such as a fibronectin-binding autolysin, an amidase/hydrolase and a holin were encoded in the viral genomes. Several ORFs encoded proteins that gave BLASTP matches with proteins from Clostridium spp. and other Gram-positive bacterial and bacteriophage genomes as well as unknown putative Collinsella aerofaciens proteins. Proteomics analysis of the purified viruses resulted in the identification of the putative pre-neck/appendage protein and a minor structural protein encoded by large open reading frames. Variants of the portal protein were identified, and several mycobacteriophage gp6-like protein variants were detected in large amounts relative to other virion proteins. The predicted amino acid sequences of the pre-neck/appendage proteins had major differences in the central portion of the protein between the two phage gene products. Based on phylogenetic analysis of the large terminase protein, these phages are predicted to be pac-type, using a head-full DNA packaging strategy.
PMCID: PMC4127328  PMID: 20963614

Results 1-25 (581425)