Restriction endonuclease map of corynebacteriophage omega ctox+ isolated from the Park-Williams no. 8 strain of Corynebacterium diphtheriae.

The toxigenic corynebacteriophage omega tox+ was isolated from the hypertoxigenic Park-Williams no. 8 (PW8) strain of Corynebacterium diphtheriae and compared with the toxigenic corynebacteriophage beta tox+. The physical size and host range of both phages were found to be identical. An endonuclease restriction map of omega tox+ was constructed, and the locations of the cohesive ends (cos), phage attachment site (attP), and the diphtheria tox operon were identified. The genome of omega tox+ was found to differ from that of beta tox+ in three regions. In addition, omega tox+ was shown to be integrated into two nontandem corynebacterial phage attachment sites (attB1, attB2) in the PW8 chromosome. The differences in the restriction endonuclease digestion maps of omega tox+ and beta tox+ and the contribution of double lysogeny are discussed in relation to the hypertoxigenicity of the PW8 strain.     

Defective site-specific integration elements are present in the genome of virulent bacteriophage LL-H of Lactobacillus delbrueckii.

The phage attachment site, attP, and the integrase-encoding gene, int, are sufficient to promote site-specific integration of the temperate phage mv4 genome into the chromosome of the Lactobacillus delbrueckii host (L. Dupont, B. Boizet-Bonhoure, M. Coddeville, F. Auvray, and P. Ritzenthaler, J. Bacteriol. 177:586--595, 1995). The mv4 genome region containing these elements was compared at the nucleotide and amino acid levels with that of the closely related virulent phage LL-H. Complex DNA rearrangements were identified; a truncated integrase gene and two sites homologous to the mv4 attP site were detected in the genome of the virulent phage LL-H. These observations suggest that the two phages derive from a common temperate ancestor.     

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.     

Staphylococcus aureus bacteriophages mediating the simultaneous lysogenic conversion of beta-lysin, staphylokinase and enterotoxin A: molecular mechanism of triple conversion

A new group of serotype F bacteriophages of Staphylococcus aureus has been found which mediates the simultaneous triple-lysogenic conversion of enterotoxin A, staphylokinase and beta-lysin. The phages were recovered fro methicillin-resistant strains of S. aureus isolated in Irish hospitals between 1971 and 1988 and from strain PS42-D, which has been used as the propagating strain for the S. aureus typing phage 42D since before 1965. The molecular mechanism of triple conversion mediated by three of these phages was determined by molecular cloning, restriction endonuclease site mapping and hybridization analysis, and compared with the mechanism of beta-lysin and staphylokinase conversion mediated by the serotype F, double-converting phase phi 13. THe genetic determinants mediating expression of enterotoxin A (entA) and staphylokinase (sak) were cloned from the DNA of the triple-converting phage and expression of the cloned determinants detected in Escherichia coli and S. aureus. The entA and sak determinants were closely linked in the phage DNA adjacent to the phage attachment site (attP) in each case and furthermore, the sak determinant of phage phi 13 was also located near its attP. The restriction maps of the entA-, sak- and attP-containing DNA regions of the three triple-converting phages were very similar to each other and to the corresponding sak- and attP- containing DNA region of phage phi 13. Hybridization analysis using a cloned beta-lysin determinant (hlb) and cloned attP-containing DNA fragments as probes demonstrated that beta-lysin conversion mediated by the triple-converting phages and phage phi 13 was caused by insertional inactivation of the chromosomally encoded hlb determinant by orientation-specific integration of phage DNA following lysogenization.     

Detection of homology to the beta bacteriophage integration site in a wide variety of Corynebacterium spp.

In toxigenic conversion of Corynebacterium diphtheriae C7, beta bacteriophage DNA integrates into either of two chromosomal attachment sites, attB1 or attB2. These attB sites share a 96-base-pair sequence with the attP sites of beta-related phages. The distribution of attB-related sites in other species of Corynebacterium was assessed by hybridization with a DNA probe containing both attB sites of the C7 strain and a second DNA probe containing the attP site of a beta-related phage. All but one of the 15 C. diphtheriae strains tested, regardless of origin or colonial type, contained at least two BamHI fragments that hybridized strongly to both of these probes under conditions of high stringency. Strains of C. ulcerans and C. pseudotuberculosis, species in which conversion to toxinogeny has also been demonstrated, also had one or two hybridizing BamHI fragments. The functionality of these sites as integration sites was demonstrated by isolating lysogens of all three species following single infection with one or more beta-related phages. As predicted, following lysogenization one of the DNA fragments that had exhibited homology with the attB1-attB2 probe was replaced by two hybridizing fragments. Other species of Corynebacterium, including pathogens and nonpathogens from animals, plant pathogens, and soil isolates also carried at least one BamHI fragment that hybridized with the attB1-attB2 and attP probes. The data indicate that sequences homologous to the beta phage integration sites in C. diphtheriae have been conserved in members of the genus Corynebacterium.     

Restriction endonuclease mapping of bacteriophage phi105 and closely related temperate Bacillus subtilis bacteriophages rho10 and rho14.

Cleavage maps of the three similar Bacillus subtilis temperate bacteriophages, phi105, rho10, and rho14, were constructed by partial digestion analysis utilizing the restriction endonuclease EcoRI. Comparison of the topography of these maps indicates that all phage DNAs posses cohesive ends and a number of EcoRI restriction sites; the fragments are conserved, and the estimated base substitution/nucleotide divergence between these phages is 0.03 to 0.07 based on conserved fragments or between 0.03 and 0.11 based on conserved cleavage sites. These lines of evidence indicate that phi105, rho10, and rho14 are closely related. Double-enzyme digestion analysis reveals that rho14 DNA has unique SalGI and BglII restriction sites and phi105 DNA has a unique SalGI restriction site, making these phages possible cloning vectors for B. subtilis.     

MM1, a temperate bacteriophage of the type 23F Spanish/USA multiresistant epidemic clone of Streptococcus pneumoniae: structural analysis of the site-specific integration system

We have characterized a temperate phage (MM1) from a clinical isolate of the multiply antibiotic-resistant Spanish/American 23F Streptococcus pneumoniae clone (Spain(23F)-1 strain). The 40-kb double-stranded genome of MM1 has been isolated as a DNA-protein complex. The use of MM1 DNA as a probe revealed that the phage genome is integrated in the host chromosome. The host and phage attachment sites, attB and attP, respectively, have been determined. Nucleotide sequencing of the attachment sites identified a 15-bp core site (5'-TTATAATTCATCCGC-3') that has not been found in any bacterial genome described so far. Sequence information revealed the presence of an integrase gene (int), which represents the first identification of an integrase in the pneumococcal system. A 1.5-kb DNA fragment embracing attP and the int gene contained all of the genetic information needed for stable integration of a nonreplicative plasmid into the attB site of a pneumococcal strain. This vector will facilitate the introduction of foreign genes into the pneumococcal chromosome. Interestingly, DNAs highly similar to that of MM1 have been detected in several clinical pneumococcal isolates of different capsular types, suggesting a widespread distribution of these phages in relevant pathogenic strains.     

A profile of native integration sites used by φC31 integrase in the bovine genome

The Streptomyces phage φC31 integrase can efficiently target attB-bearing transgenes to endogenous pseudo attP sites within mammalian genomes.To better understand the activity ofφC31 integrase in the bovine genome,DNA sequences of 44 integration events were analyzed,and 32 pseudo attP sites were identified.The majority of these sites share a sequence motif that contains inverted repeats and has similarities to wild-type attP site.Genomic DNA flanking these sites typically contained repetitive sequence elements,such as short and long interspersed repetitive elements.These sequence features indicate that DNA sequence recognition plays an important role in guidingφC31-mediated site-specific integration.In addition,BF27 integration hotspot sites were identified in the bovine genome, which accounted for 13.6%of all isolated integration events and mapped to an intron of the deleted in liver cancer 1(DLC1) gene.Also we found that the pseudo attP sites in the bovine genome had other features in common with those in the human genome.This study represents the first time that the sequence features of pseudo attP sites in the bovine genome were analyzed.We conclude that this site-specific integrase system has great potential for applied modifications of the bovine genome.     

Restriction endonuclease map of the nontoxigenic corynephage gamma c and its relationship to the toxigenic corynephage beta c.

Clear-plaque-forming mutant gamma tox- corynephages were isolated independently from nontoxigenic lysogenic Corynebacterium diphtheriae strains C7s(gamma tox-) and C4(gamma tox-). A physical map was constructed by using restriction endonucleases BamHI, EcoRI, HindIII, and KpnI. A comparison of nontoxigenic gamma c with toxigenic corynephage beta c revealed large areas of homology, including common regions for cohesive ends (cos) and attachment sites (att). Localization of the att sites on the beta prophage and correlation of the physical and genetic maps defined the orientation of the diphtheria tox operon. Diphtheria tox sequence homologies were mapped on gamma c by hybridizing 32P-labeled diphtheria tox mRNA to restriction fragments of gamma c DNA. Two regions of heterogeneity between phages beta c and gamma c were localized and these regions accounted for the 3-kilobase larger molecular size of gamma c compared with beta c. One change occurs near the tox promoter and may explain the nontoxigenic phenotype of corynephage gamma tox-.     

Detection and physical map of a omega tox+-related defective prophage in Corynebacterium diphtheriae Belfanti 1030(-)tox-.

A library of chromosomal DNA from Corynebacterium diphtheriae Belfanti 1030(-)tox- was cloned in the lambda phage vector EMBL4 and screened for sequences homologous to corynephage omega tox+ and the attB1-attB2 region of the C7(-)tox- chromosome. Two portions of the 1030(-)tox- chromosome, 35 and 30.5 kilobases long which contain, respectively, the entire region homologous to corynephage omega tox+ and the attB1-attB2 sites, were mapped with the restriction endonucleases BamHI and EcoRI. Chromosomal DNA from 1030(-)tox- was shown to contain a 15.5-kilobase region that was homologous to ca. 42% of the corynephage omega tox+ genome. These sequences were found to hybridize to three regions of the phage genome and do not contain either the diphtheria tox operon or the attP site. These sequences are distant from the chromosomal region that contains the attB1-attB2 sites. Moreover, unlike other known defective prophages, the physical map of this prophage starts at the cos site and is colinear with the vegetative phage map. The 30.5-kilobase region of the 1030(-)tox- chromosome, which contains the attB1-attB2 sites, has a central core region that is almost identical to the corresponding region of the C7(-)tox- chromosome; however, the flanking sequences in these two strains of C. diphtheriae are different.     

Genome organization of Sp beta c2 bacteriophage carrying the thyP3 gene

Thymine auxotrophs of Bacillus subtilis strains lysogenic for temperate bacteriophage SP beta c2 were transformed to prototrophy by DNA from related phage phi 3T. During transformation, the phi 3T-encoded thymidylate synthetase gene, thyP3, became integrated into the extreme right end of the SP beta c2 prophage near the bacterial citK gene. Upon heat induction, the transformed B. subtilis cells released SP beta c2T phages that could lysogenize thymine auxotrophs and convert them to prototrophy. Comparison of restriction endonuclease fragments of DNAs from SP beta c2 and SP beta c2T phages revealed that the latter contained a large region of deletion and substitution near the center of the chromosome. This region included the phage attachment site on the SP beta c2 genome.     

Characterization of XerC- and XerD-dependent CTX phage integration in Vibrio cholerae

CTXphi is a filamentous bacteriophage that encodes cholera toxin and integrates site-specifically into the larger of the two Vibrio cholerae chromosomes. The CTXphi genome lacks an integrase; instead, its integration depends on the chromosome-encoded tyrosine recombinases XerC and XerD. During integration, recombination occurs between regions of homology in CTXphi and the V. cholerae chromosome. Here, we define the elements on the phage genome (attP) and bacterial chromosome (attB) required for CTXphi integration. attB is a short sequence composed of one binding site for XerC and XerD spanning the site of recombination. Together, XerC and XerD bind to two sites within attP. While one XerC/D binding site in attP spans the core recombination region, the other site is approximately 80 bp away. Although integration occurs at the core XerC/D binding site in attP, the second site is required for CTXphi integration, suggesting it performs an architectural role in the integration reaction. In vitro cleavage reactions showed that XerC and XerD are capable of cleaving attB and attP sequences; however, additional cellular processes such as DNA replication or Holliday junction resolution by a host resolvase may contribute to integration in vivo.     

Superinfection exclusion by heteroimmune corynebacteriophages.

Superinfection of Corynebacterium diphtheriae C7(beta) by heteroimmune phage gamma is productive, whereas superinfection by gamma-bin mutants is for the most part nonproductive. Exclusion of gamma-bin phage occurred after its DNA had penetrated and was partially expressed in the heteroimmune lysogen. All of the infected cells were killed, and lysis was observed. The beta inhibitor causing exclusion was produced during the prophage state and appeared to be distinct from immune repressor. The ability of gamma-bin phage to superinfect C7(beta) productively could be restored by recombination with beta phage, indicating that both beta and gamma phages contain either indentical or similar alleles of the bin gene. The bin gene was mapped by vegetative and prophage crosses and found to be located in the region of the phage genome concerned with regulation. Both beta and gamma wild-type phages induced the resident prophage in a significant fraction of superinfeted heteroimmune lysogens. This, coupled with the fact that induction of C7(beta) abolished exclusion, suggests that the bin gene product acts as antirepressor, i.e., it reduces the level of heteroimmune repressor either directly or indirectly. The gamma-bin mutants either failed to produce antirepressor or did so with reduced efficiency. Antirepressor activity was negatively controlled by homoimmune repressor. The isolation of beta mutants that appeared bin-like suggests that beta and gamma phages contain homologous systems of exclusion and antiexclusion. Exclusion of gamm-bin by beta phage in gram-positive C. diphtheriae exhibited striking parallels to the sieB exclusion described for phages P22 and lambda in gram-negative organisms. The extended similarities of these coryngephages to lambda bacteriophage is noted.     

Phage R4 integrase mediates site-specific integration in human cells.

The R4 integrase is a site-specific, unidirectional recombinase derived from the genome of phage R4 of Streptomyces parvulus. Here we define compact attB and attP recognition sites for the R4 integrase and express the enzyme in mammalian cells. We demonstrate that R4 integrase functions in human cells, performing efficient and precise recombination between R4 attB and attP sites cloned on an extrachromosomal vector. We also provide evidence that the enzyme can mediate integration of an incoming plasmid bearing an attB or attP site into endogenous sequences in the human genome. Furthermore, when R4 attB and attP sites are placed into the human genome, either by random integration or at a specific sequence by using the phi C31 integrase, they act as targets for integration of incoming plasmids bearing R4 att sites. The R4 integrase has immediate utility as a site-specific integration tool for genome engineering, as well as potential for further development.     

Using phiC31 integrase to make transgenic Xenopus laevis embryos

Bacteriophage phiC31 produces the enzyme integrase that allows the insertion of the phage genome into its bacterial host. This enzyme recognizes a specific DNA sequence in the phage (attP) and a different sequence in the bacterium (attB). Recombination between these sites leads to integration in a reaction that requires no accessory factors. Seminal studies by the Calos laboratory demonstrated that the phiC31 integrase was capable of integrating plasmid with an attB site into mammalian genomes at sites that approximated the attP site. We describe the use of attB-containing plasmids with insulated reporter genes for the successful integration of DNA into Xenopus embryos. The method offers a way to produce transgenic embryos without manipulation of sperm nuclei using microinjection methods that are standard for experiments in Xenopus laevis. The method aims to allow the non-mosaic controlled expression of new genetic material in the injected embryo and compares favorably with the time that is normally taken to analyze embryos injected with mRNAs, plasmids, morpholinos or oligonucleotides.     

Genetic analysis of chromosomal regions of Lactococcus lactis acquired by recombinant lytic phages

Recombinant phages are generated when Lactococcus lactis subsp. lactis harboring plasmids encoding the abortive type (Abi) of phage resistance mechanisms is infected with small isometric phages belonging to the P335 species. These phage variants are likely to be an important source of virulent new phages that appear in dairy fermentations. They are distinguished from their progenitors by resistance to Abi defenses and by altered genome organization, including regions of L. lactis chromosomal DNA. The objective of this study was to characterize four recombinant variants that arose from infection of L. lactis NCK203 (Abi(+)) with phage phi31. HindIII restriction maps of the variants (phi31.1, phi31.2, phi31.7, and phi31.8) were generated, and these maps revealed the regions containing recombinant DNA. The recombinant region of phage phi31.1, the variant that occurred most frequently, was sequenced and revealed 7.8 kb of new DNA compared with the parent phage, phi31. This region contained numerous instances of homology with various lactococcal temperate phages, as well as homologues of the lambda recombination protein BET and Escherichia coli Holliday junction resolvase Rus, factors which may contribute to efficient recombination processes. A sequence analysis and phenotypic tests revealed a new origin of replication in the phi31.1 DNA, which replaced the phi31 origin. Three separate HindIII fragments, accounting for most of the recombinant region of phi31.1, were separately cloned into gram-positive suicide vector pTRK333 and transformed into NCK203. Chromosomal insertions of each plasmid prevented the appearance of different combinations of recombinant phages. The chromosomal insertions did not affect an inducible prophage present in NCK203. Our results demonstrated that recombinant phages can acquire DNA cassettes from different regions of the chromosome in order to overcome Abi defenses. Disruption of these regions by insertion can alter the types and diversity of new phages that appear during phage-host interactions.     

Phage-conversion of cytotoxin production in Pseudomonas aeruginosa

We isolated a temperate phage which carried the cytotoxin gene (ctx) from a cytotoxin (CTX)-producing Pseudomonas aeruginosa strain, PA158. The phage, phi CTX, had a head with a hexagonal outline and a contractile tail with tail fibres. The phage genome was a linear double-stranded 35.5 kb DNA with single-stranded cohesive ends (cos). The attP, cos and ctx genes were all located very close to one another within a 2.3 kb segment on the phage genome in the order given (in the circular form). phi CTX converted CTX non-producing P. aeruginosa strains into CTX producers. A single copy of phi CTX DNA was integrated at the same site on the host chromosome (attB) in every lysogen, including PA158. However, the amount of CTX produced in these lysogens varied from strain to strain and was less than that in PA158.