Integration of corynebacteriophages beta tox+, omega tox+, and gamma tox- into two attachment sites on the Corynebacterium diphtheriae chromosome.

The bacterial attachment sites of independently isolated Corynebacterium diphtheriae strains C7s and (belfanti)1030 lysogenic for corynebacteriophages beta tox+, omega tox+, and gamma tox- were determined by Southern blot analysis. Both corynebacterial strains contained two distinct bacterial attachment sites (attB1 and attB2). We found that infection by any of the three closely related corynebacteriophages may give rise to single, double, and triple lysogens. In the case of toxigenic C. diphtheriae strains C7s(beta tox+) and C7s(omega tox+), the final yields of diphtheria toxin produced under optimal conditions were equivalent and varied by one-, two-, or threefold depending upon the number of integrated prophage.     

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.     

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 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.     

Physical map of the chromosomal region of Corynebacterium diphtheriae containing corynephage attachment sites attB1 and attB2.

The chromosome of Corynebacterium diphtheriae C7 was recently shown to contain two equivalent attachment sites (attB1 and attB2) for lysogenization by corynephages (R. Rappuoli, J.L. Michel, and J.R. Murphy, J. Bacteriol. 153:1202-1210, 1983). Portions of bacterial chromosome containing each attB site, as well as a 3.5-kilobase (kb) EcoRI fragment containing both attB1 and attB2 sites, were cloned in the pUC8 plasmid vector. Restriction endonuclease mapping and Southern blot hybridization analysis of restriction endonuclease fragments showed that attB1 and attB2 are 2.25 kb apart on the chromosome. Furthermore, a 0.85-kb HincII-EcoRI restriction endonuclease fragment containing attB1, a 0.77-kb HincII-BamHI fragment containing attB2, and a 1.2-kb EcoRI-BamHI fragment containing attP share short homologous regions. No homology was detected between the sequences flanking the two attB sites. The isolation of a segregant which had lost the entire chromosomal segment contained between attB1 and attB2 suggests that this region is not essential for growth.     

Isolation and characterization of extragenic suppressor strains of Corynebacterium diphtheriae.

The isolation and characterization of two different nonsense suppressor strains of Corynebacterium diphtheriae C7 sup+(-)tox- are described. Appropriate lysogens of these strains with corynephage beta, carrying known class II tox premature polypeptide chain termination mutations [C7sup-1(betatox-30) and C7sup-2(betatox-45)], each produce a 62,000-dalton polypeptide with nicotinamide adenine dinucleotide: elongation factor-2 adenosine diphosphate ribosyltransferase activity in addition to a chain-terminated polypeptide of 30,000 or 45,000 daltons, respectively. In addition, purified protein of 62,000 daltons, resulting from the suppression of the nonsense mutations tox-30 and tox-45, will react with antisera purified against the terminal 17,000 daltons of the toxin molecule and are immunologically identical to toxin by radial immunodiffusion. The suppression pattern of lysogenic derivatives of C7sup-1(-)tox- and C7sup-2(-)tox- with other class II and III mutants of corynephage beta was determined.     

DNA homology study of Corynebacterium diphtheriae v. gravis groups I, II and III, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis (ovis)

The homology of genomes within Krylova 's groups I, II and III of C. diphtheriae, including toxigenic C. diphtheriae and their nontoxigenic precursors within the same group, was confirmed by the method of DNA/DNA molecular hybridization; the homology of DNA within the groups was 89-103%, the thermostability of heteroduplexes being high (on the level of homoduplexes ). The heterogeneity of genomes within these 3 groups of cultivar gravis was confirmed, which made it possible to consider C. diphtheriae, groups I, II and III, to belong to different, though closely related species; in intergroup hybridization the homology of DNA varied, as a rule, between 66% and 73%, while the thermostability of heteroduplexes was low: delta T50 was -3 degrees C to -6 degrees C. The differences in genomes (on the level of different species) between 3 groups of C. diptheriae v. gravis on one hand and C. diphtheriae v. mitis C7 (-) tox- and its convertant C7 (beta) tox+ of phage tox+ on the other hand (DNA homology being 56-62%), as well as between C. diphtheriae v. intermedius No. 328 tox+ on one hand and the representatives of 3 groups of C. diphtheriae v. gravis and C. diphtheriae v. mitis, strain C7 (beta) tox+, on the other hand (DNA homology being 42-43%) were revealed. The heterogeneity of genomes (on the level of different genera) was revealed between C. diphtheriae strains, cultivars gravis (groups I, II and III), mitis (C7(-) tox- and C7 (beta) tox+) and intermedius (No. 328 tox+) on one hand and C. ulcerans and C. pseudotuberculosis (ovis) strains on the other hand; DNA homology was 11-17% for C. ulcerans and 22-26% for C. pseudotuberculosis (ovis), the thermostability of heteroduplexes being at the lowest level (delta T50 was -11 degrees C to -13 degrees C). As a result, C. diphtheriae, classified by Bergey as a single species, was found to comprise 5 species detected by means of marking in accordance with their phenotypical features and genome structure, carried out by the method of DNA/DNA molecular hybridization; among these species were group I, II and III strains of cultivar gravis, strain C7 of cultivar mitis and strain No. 328 of cultivar intermedius. C. ulcerans and C. pseudotuberculosis (ovis) strains investigated in this study can possibly be placed outside the genus including 5 C. diphtheriae species.     

Pangenomic study of Corynebacterium diphtheriae that provides insights into the genomic diversity of pathogenic isolates from cases of classical diphtheria, endocarditis, and pneumonia

Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae. Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ω(tox)(+) phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox(+) corynephage. DNA binding sites of the tox-controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.     

Patterns of lambda Int recognition in the regions of strand exchange

Int protein has two classes of binding sites within the phage att site: the arm-type recognition sequences are found in three specific sites that are distant from the region of strand exchange; the junction-type recognition sequences occur as inverted pairs around the crossover region in both attP and attB. During recombination between attP and attB each of the four DNA strands is cut at a homologous position within each of the junction-type Int binding sites. In all four junction-type sites Int protein interacts primarily with the same face of the DNA helix, as determined by those purine nitrogens that are protected against methylation by dimethylsulfate. Efficient secondary attachment sites for lambda contain sequences with partial homology to the junction-type binding sites. In addition, the sequence between, but not part of, the two junction-type sites (the overlap region) is strongly conserved in secondary att sites. Thus, in the vicinity of strand exchange, attP and a recombining partner, such as attB, are very similar; each comprises two junction-type Int recognition sites and an overlap (crossover) region.     

Comparative studies with tox plus and tox minus corynebacteriophages

The characteristics of nine inducible temperate corynebacteriophages designated alpha(tox+), beta(tox+), P(tox+), gamma(tox-), pi(tox+), K(tox-), rho(tox-), L(tox+), and delta(tox+) have been compared. Virion morphology and ability to recombine genetically with the well-studied phage beta(tox+) have been correlated with other properties of the phages, and the distribution of the genetic marker tox+ among related and relatively unrelated corynebacteriophages has been analyzed. The immunity specificity, host range, and plaque morphology of each phage were determined. The phages can be separated into five groups with different immunity specificities. Each type of host range previously recognized in mutants of phage beta(tox+) was present in one or more of the phages included in the present study, and the phages were found to produce plaques of several different morphological types. Representative phages with each of the five types of immunity specificity were further characterized with respect to virion morphology, ability to recombine with phage beta(tox+), latent period, average burst size, and neutralization by homologous and heterologous antiphage sera. All of these phages have polyhedral heads and long slender tails, but two distinct morphological types were distinguished by the sizes and proportions of the components of the virions. Only phages of the same morphological type as beta(tox+) were capable of genetic recombination with beta(tox+), but morphological similarity between phages was not sufficient to insure interfertility. The phages which recombined with beta(tox+) resembled one another in plaque morphology, latent period, and average burst size, whereas phages which failed to recombine with beta(tox+) differed in these characteristics. The phages capable of genetic recombination with beta(tox+) were found to differ from each other in immunity specificity, host range, neutralization by antiphage sera, and toxinogenicity. Thus, these latter characteristics are of limited value in establishing the extent of relatedness between corynebacteriophages. The genetic marker tox+ was not consistently correlated with any other property of the corynebacteriophages analyzed in this study. The most striking finding regarding the distribution of the tox+ marker is its presence both in beta(tox+) and delta(tox+), phages which fail to recombine genetically and which differ in virion morphology. The presence of the tox+ marker in genetically unrelated corynebacteriophages poses many questions concerning the origin(s) of tox+ and the evolution of the phage-host interactions which determine the ability of corynebacteria to synthesize diphtherial toxin.     

Plasmids carrying cloned fragments of RF DNA from the filamentous phage φLf can be integrated into the host chromosome via site-specific integration and homologous recombination

Different regions of RF DNA from the filamentous bacteriophage phiLf were cloned in Escherichia coli vectors that can not be maintained in Xanthomonas. After introduction into X. campestris pv. campestris 17 (Xc17), most of these constructs were found to integrate into the host chromosome, either by recA-dependent homologous recombination or recA-independent site-specific integration. Mutations in himA, which codes for the alpha-subunit of the Integration Host Factor, does not affect the integration. Integration occurs into a chromosomal region which harbors a copy of a defective phage (4445 bp) that shares a high degree of identity with the phiLf genome. While various parts of the 4445-bp region are susceptible to homologous recombination, site-specific integration requires the attB sequence on the chromosome and the phage attP. The attB shows a high level of sequence identity (22 out of 28 bp) to the dif site required for E. coli Xer site-specific recombination, including the 6-bp central region, and 8/11 identity in both the left XerC-binding arm and the right XerD-binding arm, with the innermost 5 nt of the arms forming a dyad symmetry that is also present in dif. The attP has the same central region and shows 10/11 identity to the dif site in the left arm, but the sequence of the right arm is less conserved than that of attB. The smallest regions still capable of mediating integration are a cloned 72-bp phiLf attP-containing sequence and a 51-bp Xc17 attB-containing sequence, which was reinserted into the Xc17 chromosome after the 4445-bp region had been deleted, indicating that accessory sequences are not necessary and that the integrase required for site-specific integration is neither specified by the 4445-bp Xc17 chromosomal region nor encoded by the phiLf genome.     

Genetic Analysis of tox+ and tox? Bacteriophages of Corynebacterium diphtheriae

A series of mutants derived from the temperate corynebacteriophages beta(tox+), gamma(tox-), and L(tox+) was isolated and characterized. In three-factor crosses between mutant beta phages the relative map order of the genetic markers determining extended host ranges (h and h') and loss of ability to lysogenize (c) was found to be h--c--h'. Recombination between markers was observed in matings between phage beta and the heteroimmune corynebacteriophages gamma and L. In such matings between heteroimmune phages the c markers of phages beta and gamma failed to segregate from the imm markers which determine the specificity of lysogenic immunity in these phages. The factor which directs the synthesis of diphtherial toxin during infection of appropriate corynebacterial hosts by toxinogenic corynebacteriophages is designated tox(+). It was possible to show that the tox(+) determinant of phage beta behaves as a single genetic element which occupies a position between the loci h and imm on the genetic map of this phage. Genetic recombination between mutants of phage beta occurred at very low frequencies in biparental matings performed by mixed infection of Corynebacterium diphtheriae C7(s)(-)(tox-). Considerably higher recombination frequencies were observed when lysogenic bacterial strains carrying one parental phage as prophage were induced by ultraviolet irradiation and then superinfected by the second parental phage. Maximal stimulation of genetic recombination between mutant beta phages was detected when superinfection followed ultraviolet irradiation of the lysogenic cells within a limited period of time. In matings between phages with incomplete genetic homology, the stimulation of recombination by ultraviolet radiation was much less effective.     

System for the Investigation of the Bacteriophage-directed Synthesis of Diphtherial Toxin

Lytic corynebacteriophage betahv64(tox+) has been characterized, and methods for studying the expression of its tox(+) gene in nontoxinogenic Corynebacterium diphtheriae strain C7(s)(-)(tox-) described. During one cycle of viral growth there was a 1 million-fold increase in extracellular toxin. Both the conditions of the experiment and the use of purified phage, free from toxin, support the conclusion that all of the toxin was newly formed. This toxin was immunochemically indistinguishable from standard toxin produced by the PW8(r)(Pdi)(tox+) strain. Chloramphenicol was found to be an effective agent for synchronizing the initiation of viral growth. Once chloramphenicol was removed, intracellular toxin appeared and continued to increase throughout the latent period. Proflavine, added early in the latent period, blocked phage maturation without similarly affecting yields of toxin. Iron exerted a limited inhibitory effect on final toxin levels attained.     

Regulation of toxinogenesis in Corynebacterium diphtheriae. I. Mutations in bacteriophage beta that alter the effects of iron on toxin production

Diphtherial toxin is produced in maximal yields by Corynebacterium diphtheriae (C7(beta tox+) only when iron is present in growth-limiting amounts. Toxin production is markedly decreased under high-iron conditions. We studied the role of the bacteriophage beta genome in this apparent regulation of toxin production by iron. Using a passive immune hemolysis assay to detect toxin antigen production in individual plaques, we identified rare phage mutants that were toxinogenic in high-iron medium. Lysogenic derivatives of C. diphtheriae C7 harboring such phage mutants were constructed. The lysogens were compared with wild-type strain C7(beta) for their ability to produce toxin in deferrated liquid medium containing varying amounts of added iron. Quantitative tests for extracellular toxin were performed by competitive-binding radioimmunoassays. We identified phenotypically distinct mutant strains that produced slightly, moderately, or greatly increased yields of toxin antigen under high-iron conditions. The toxin produced by the mutant lysogens was biologically active and immunochemically indistinguishable from wild-type toxin. Complementation experiments demonstrated that the phage mutation designated tox-201 had a cis-dominant effect on the expression of the toxin structural gene of phage beta. The characteristics of the tox-201 mutation suggest that it defines a regulatory locus of phage beta that is involved in control of toxinogenesis by iron in C. diphtheriae.     

Comparative analysis of the DNA of Corynebacterium diphtheriae phages and the cloning of the gene determining diphtheria toxin synthesis

The analysis of the DNA of one nontoxigenic C. diphtheriae phage and two toxigenic ones has revealed that phage phi 984tox+ belongs to omega-like tox+ phages, phage phi 9tox+ is a representative of a new group of phages and phage B (Freeman) tox is a deletion mutant of phage beta. The location of this deletion on the physical map of this phage has been established. To obtain the physical map of phage phi 984tox+, the complete library of internal DNA fragments has been constructed in vector pBR 322. The gene of native diphtheria toxin has been cloned in vectors pBR 322 and pUR 250. Plasmids pUR 250 with the inserts of the toxin gene have been shown to be unstable if tox and lac promoters are located in tandem before the body of the toxin gene. The prolonged cultivation of clones having such structure leads to the formation of a spontaneous mutation located in the region coding the C-end part of the A-fragment of the toxin.     

Orientation of the tox gene in the prophage of corynebacteriophage beta.

The orientation of the gene for diphtheria toxin, tox, in the prophage of converting corynebacteriophage beta has been determined. The orientation of tox in prophage and that reported simultaneously by Holmes (1976) for vegetative phage are compatible with the hypothesis that beta phage is inserted into the chromosome of its bacterial host by means of a mechanism similar to that described for lambda phage, and that the phage attachment site lies between the tox and imm genes. The position of three tox mutations that are phenotypically CRM- has also been determined. Relative to the tox-45 mutation, they are located more proximally to the end of the tox structural gene that corresponds to the amino terminal of diphtheria toxin.