[Cloning in Escherichia coli of the mutant gene coding the diphtheria toxin]. / Klonirovanie v Escherichia coli mutantnogo gena, kodiruiushchego difteriinyi toksin.

Bacteriophage beta 45 of Corynebacterium diphtheriae was harvested. The extracted DNA of the bacteriophage was digested by the restriction endonuclease BamHI and inserted into the BamHI cleavage site of pUC19 vector plasmid. Plasmid pNVY5 containing a mutant gene crm45 of diphtheriae toxin in a 3.9 bpn fragment was isolated from the hybrid plasmids obtained. Cell free extracts of E. coli strain TG1 (pVNY5) contain the nontoxic protein crm45 possessing the specific enzymatic activity of diphtheriae toxin (ADP ribosylation on wheat elongation factor two). According to orientation of BamHI fragment in pNVY5 plasmid it is concluded that the crm45 gene is expressed using its own promoter.

[Comparison of the physicochemical structure of DNA in Corynebacterium related to C. diphtheriae v. mitis, intermedius and gravis]. / Sravnenie fiziko-khimicheskoi strucktury DNK Corynebacterium, otnoshiykh k C. diphtheriae v. mitis, intermedius i gravis.

The hybridization of DNA of 19 C. diphtheriae v. mitis strains with DNA of 3H-labeled C. diphtheriae v. mitis strain C7(beta) tox+ resulted in revealing four homology levels: I (102-98%), II (78-68%), III (61-50%) and IV (42-25%). Homology levels I and II differed from one another on the level of species. In the hybridization of DNA of the same strains with DNA of 3H-labeled C. diphtheriae v. gravis (group II) strain 165(1) tox+ three homology levels were revealed; levels I and II differed from one another on the level of the genus. In the first and second series of the experiments the distribution of DNA of the above strains on these levels not always coincided. The genomes of C. diphtheriae v. mitis strains 7111 tox+ and 132 tox+ were shown to differ from those of both reference strains on the level of the genus. In strains C7(beta) tox+ and PW-8 tox+ Massachusetts the genetic similarity of their genomes on the level of species was disclosed. Among C. diphtheriae v. mitis and intermedius strains at least 5 homology levels were revealed; this fact suggests the presence of at least 5 bacterial species among them. The relationship of strains on the level of species within groups I and II of C. diphtheriae v. gravis was confirmed: DNA of the related strains belonging to the corresponding group were always to be found within the same homology level. Also confirmed was the fact that the strains of groups I and II, as well as strain C7(beta) tox+, differed on the level of species.(ABSTRACT TRUNCATED AT 250 WORDS)

[DNA homology study of Corynebacterium diphtheriae v. gravis groups I, II and III, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis (ovis)]. / Izuchenie gomologii DNK grupp I, II i III Corynebacterium diphtheriae v. gravis, Corynebacterium ulcerans i 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.

[Improvement of the preliminary classification of Corynebacterium diphtheriae v. gravis]. / Utochnenie predvaritel'noi skhemy podrazdeleniia vozbuditelei difterii, otnosimykh k C. diphtheriae v. gravis.

In this study the previously published preliminary scheme for the subdivision of toxigenic and nontoxigenic Corynebacterium diphtheriae, classified with cultivar gravis, is made more precise. 3 groups remain in this scheme: I, II and III; each of them contains toxigenic C. diphtheriae (subgroup a) and nontoxigenic precursors of C. diphtheriae (subgroup b). For the first time nontoxigenic analogs of C. diphtheriae, phagovar OPQSTg, have been introduced into group I and newly discovered toxigenic C. diphtheriae, phagovar K, with their nontoxigenic precursors converted by phages 5 tox+, 6 tox+ and W tox+ have been introduced into group III. Group IV has been provisionally excluded from the scheme because this group comprises a small number of strains (3 strains). This classification can already be used in research practice for a finer differentiation of strains classified with cultivar gravis and for correct epidemic orientation.

[The species C. diphtheriae]. / O vide C. diphtheriae.

Corynebacteria (C. diphtheriae var. gravis, mitis, minimus, intermedius, C. belfanti, C. ovis, C. ulceraus) producing diphtheria toxin are found as variants sharply differing from one another morphologically (from long branching rods to coccobacilli), culturally, biochemically and by other signs. The toxin synthesis in them was determined by genetically heterogeneous prophages tox+; tox+ virus exchange is possible between the corynebacteria. Since production of specific toxin, the cardinal sign of C. diphtheriae species, is connected with the genoms of genetically heterogeneous viruses tox+, the circle of whose hosts is very wide, it can be supposed that there is no genetically homogeneous taxonomic category "diphtheriae species". Several species of corynebacteria--lysogenized (toxigenic) or capable of being lysogenized (nontoxigenic) with tox+ viruses are included into the composition of the corynebacteria groups which medicine considers as "species" for over 80 years. A possiblity of becoming a diphtheria causative agent is determined by the capacity of corynebacteria to become infected with the circle of viruses tox+ the DNA of which can become stably integrated with the gene of this bacterium. The mentioned approach to taxonomy is possibly applicable to microorganisms in which the pathogenicity signs (the toxin) were determined, as in C. diphtheriae, in the virus genoms (Bac. botulinus).

[Use of the corycin test and of bacteriophage typing for genetic marking of corynebacteria with reference to C. diphtheriae of the gravis type]. / Primenenie koritsin-testa i fagotipirovaniia dlia geneticheskogo markirovaniia korinbacterii, otnosimykh k C. diphtheriae tipa gravis

Nontoxigenic C. diphtheria, fermenting starch, of convertible phage types ABCDFCH, ABCDFG, ABCDF, ABCD, ABD, ACDf, CDf, AF and A, and also strains of nonconvertible bacteriophage types I, H and K proved to be noncoricynogenic in the corycine test and weakly sensitive to bacteriocin of the No. 3463 test strain (a study was made of a total of 502 strains). Strains (383) isolated from 354 patients and carriers--starch-fermenting and toxigenic to the gravis type--were toxigenic were lysed by a combination of bacteriophages O, P, Q, R, S, T produced an active bacteriocine against the test strains No. 23 BC, and were insensitive to bacteriocine of the strain No. 3453. Strains of phage type G(g) were noncorycinogenic. Only one strain of phage type G, which produced corycine, active against the test strain No. 23 BC served as an exception. With the aid of phages A, B, C, D, F, G, H, I, K it was possible to separate 244 strains of the phage type G(g) into 3 phage types--bc, ABfGH and Abg. Strains of phage type ABfGH were noncorycinogenic; the only strain of phage type bG available produced bacteriocine.