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Intraspecies genetic differentiation of nontoxigenic strains of Vibrio cholerae of El Tor biovar containing one of the key pathogenicity genes, tcpA, is studied along with the phylogenetic relationships between these strains and toxigenic isolates. Comparative analysis of the whole genome nucleotide sequences demonstrates for the first time that ctxA – tcpA + strains vary considerably and can be clustered into two separate groups, the CTXφ–RS1φ +VPI+VSP+/CTXφ–RS1φ–VPI+VSP+ isolates and the CTXφ–RS1φ–VPI+VSP– isolates, differing in their epidemiological significance. In the course of model experiments, it is established that nontoxigenic potentially epidemic CTXφ–RS1φ +VPI+VSP+/CTXφ–RS1φ–VPI+VSP+ isolates are derivatives of toxigenic strains. The results of whole genome SNP analysis of 35 Vibrio cholerae strains confirm these data and indicate genetic remoteness of nontoxigenic CTXφ–RS1φ–VPI+VSP– strains both from the potentially epidemic strains and from the toxigenic isolates. It is found that the genomes of the CTXφ–RS1φ–VPI+VSP– strains contain unique SNPs which are characteristic of them alone. The new data on the structure of the genome of nontoxigenic strains with different epidemiological significance may be further used for their genetic differentiation.

The molecular-genetic peculiarities of the prophage CTXφ genome, the VPI-1 pathogenicity island, and the VSP-II pandemicity island in recently emerged, genetically altered Vibrio cholerae El Tor strains have been studied. The genome of the prophage CTXφ, which contains the ctxAB operon, which codes cholera toxin (CT), was shown to be unstable. A comparative analysis of the nucleotide sequences of the two phage genome regions (the ctxB gene and ctxAB operon promoter region) among 23 genovariant strains allowed us to reveal the presence of distinct ctxB gene alleles (ctxB1 or ctxB7) that differed in single-nucleotide substitutions and the polymorphism of the ctxAB operon promoter region. An analysis of the VPI-1 structure showed that tcpA gene that encodes the basic protein of the toxin-coregulated pilli (TCP) was represented in the genovariants by four alleles. The variability of the VSP-II was also revealed. It was manifested in the occurence of the deletions of varying length (2069 or 13 105 bp).

Genetic organization of 52 Vibrio cholerae El Tor biotype preseventh and seventh pandemic strains isolated in various periods was studied by PCR assay and DNA-DNA hybridization. It was established that the genome of most ancient of analyzed strains isolated from a diarrhea patient in 1910 was devoid of CTX and RS1 prophages, vibrio pathogenicity islands (VPI and VPI-2), and pandemic islands (VSP-1 and VSP-2) that contain key virulence genes. The appearance of pathogenic properties in cholera vibrios for the first time causing a local outbreak of cholera in 1937 is connected with the acquisition of VPI and CTX that carried genes tcpA and ctx-AB, respectively, which are responsible for the colonization of small intestine and encode the production of cholera toxin. The appearance of seventh pandemic agent for cholera was shown to correlate with the acquisition by its precursor of two additional blocks of genes VSP-1 and VSP-2. This finding strongly supports the involvement of these genes in formation of the pandemic potential in strains. Molecular typing methods allowed elucidation of differences in the genetic organization between prepandemic and pandemic strains. The detected variability of the genome of contemporary virulent strains may be a reason for the occurrence of etiological agent for cholera with new properties.

Two fractions (1 and 2) of the galactomannan from seeds of sophora (Styphnolobium japonicum) were isolated using cold and hot aqueous extraction with a total yield of 12.88%. The two fractions differed by the ratio between mannose (Man) and galactose (Gal) residues (4.8:1 and 5.3:1, respectively) and molecular weight (1190 and 1400 kDa, respectively). Aqueous solutions of these fractions were optically active ([alpha]D +4.80 degrees and -3.36 degrees, respectively) and highly viscous ([eta] 1028.8 and 1211.2 ml/g). 13C NMR spectra of both fractions were identical with respect to the number and positions of signals, which indicates that their primary structures were identical. Using chemical and spectroscopic (IR and NMR) methods, it was shown that the galactomannan has a main chain consisting of 1,4-beta-D-mannopyranose, some residues of which (16 and 17% in fractions 1 and 2, respectively) are alpha-galactosylated at the C-6 position. Frequencies of differently substituted mannobiose blocks in the chain, calculated for fraction 1 using NMR spectroscopic data, were 0.13 for the disubstitited blocks Gal(Man-Man)Gal, 0.37 for the sum of monosubstituted blocks Gal(Man-Man) and (Man-Man)Gal, and 0.50 for the unsubstituted block Man-Man.

New data were obtained concerning cell sensitivity of pathogenic strains of cholera vibrions, which belong to the serogroup O1 of classical biovar, to the temperate bacteriophage K139, the native host of which is Vibrio cholerae O139. Molecular-genetic and biochemical studies showed that phage 139 integrated into the chromosome of strains V. cholerae O1 can change their toxigenic properties. A change in the production of cholera toxin (CT) in lysogens is associated both with an increase in the activity of the toxR regulatory gene and with a distortion of the structure of a chromosomal DNA region that contains a copy of the operon ctxAB encoding CT biosynthesis.

Water-soluble glucomannan from roots of Eremurus zangezuricus Mikheev was studied. The polysaccharide contains D-glucose, D-mannose, and acetyl groups in the molecular ratio of 1:2.8:0.38. 13C NMR studies showed that the polysaccharide under study is a linear, partly acetylated 1,4-beta-D-glucomannan. The acetyl groups are attached to the C2 and C3 of mannopyranose units. The polymer contains, on average, one acetyl group per seven mannose units. The glucomannan of E. zangezuricus has the following parameters: [alpha]D = -37.5 degrees, [eta] = 3.73 dl/g, and Mw = 151 kDa.

Water-soluble glucomannans from roots of Eremurus iae and E. zangezuricus were studied. These polysaccharides were shown to contain 28.8; 69.0, and 2.2% (E. iae) and 22.6; 74.8, and 2.6% (E. zangezuricus) of D-glucose, D-mannose and acetyl groups, respectively. Their IR spectra were identical and revealed the presence of 1,4-beta-glycosidic bonds and ester carbonyl groups. 13C-NMR spectroscopy revealed both polysaccharides to be linear partially acetylated 1,4-beta-D-glucomannans. Acetyl groups substituted C-2- and C-3-hydroxyls of mannopyranose residues. Comparison of 13C-NMR data and the results of correlation analysis enables a conclusion to be made that acetyl groups can substitute no more than one OH-group in the mannopyranosyl residue. [alpha]D = -34.0 degrees, [eta] and molecular weights (MW) for E. iae polysaccharide were determined to be -34.0, 6.5 dl/g, and 265.5 kDa, respectively, and for E. zangezuricus polysaccharide -38.2, 5.4 dl/g, and 233.5 kDa, respectively. A correlation between intrinsic viscosities of polysaccharides and their molecular masses determined by HPLC was revealed.

The composition and structure of a galactomannan from seeds of Astragalus lehmannianus, an endemic legume species, is reported for the first time. The purified galactomannan (yield, 4.8%) contained 55% D-mannose and 45% D-galactose and had a molecular weight of 997.03 kDa. Its aqueous solutions were optically active and highly viscous (the specific rotation, [alpha]D, equaled +81.3 degrees; the characteristic viscosity, [eta], 868.4 ml/g). Chemical, chromatographic, and spectral (IR and 13C-NMR spectroscopy) methods were used to demonstrate that the main chain of the molecule is formed by residues of 1,4-beta-D-mannopyranose, 78% of which are substituted at position 6 with single alpha-D-galactopyranose. The distribution of galactose along the chain was calculated from NMR spectra: frequencies of occurrence, per pair of neighboring mannose units, of (1) two substituents, (2) one substituent, and (3) no substituents were 65.3, 31.5, and 3.2%, respectively. The specific rotation of galactomannans was shown to correlate with their content of galactose.

To study the possibilities of genetic exchange between Vibrio cholerae of O1 and non-O1 serogroups, donor and recipient strains were developed. It was shown that toxicogenic strains of V. cholerae non-O1 appeared in vitro and in vivo as the result of conjugative transfer of rfb-NAG genes from avirulent V. cholerae non-O1 strains to toxicogenic strains belonging to V. cholerae O1 classical and eltor biovars. These genes are responsible for synthesis of O antigen of non-O1 serotype. It was established that foreign rfb-NAG genes have no effect on virulence properties of a causative agent of cholera. Apparently, pathogenic V. cholerae non-O1 strains with cholera toxin genes are generated due to transfer of rfb-NAG genes under natural conditions.

Two replicons, pOX38 (in F-factor derivative lacking all IS elements) and pCT105 (containing cholera toxin operon cloned in pBR322) have been fused to produce recombinant plasmid, pCO109, which was then introduced into Vibrio cholerae eltor by conjugation. Restriction analysis showed pCO109 to dissociate in V. cholerae cells at a higher frequency than in Escherichia coli strains, its pOX38 component being lost, while the pCT105 component demonstrated relative stability. V. cholerae eltor RV79 (pCT105) produced 4-5 micrograms/ml of cholera toxin. Occasional insertion of cloned vctA, B operon into RV79 chromosome was also observed.

The results of cholera vibrio chromosomal mapping using Vibrio cholerae classica and V. cholerae eltor donor strains obtained with the help of various R. plasmids, are summarized in the paper. A genetic map of V. cholerae chromosome was established showing the order of 35 gene markers. The relationship between the genetic structures of cholera eltor and classical vibrio biotypes is discussed.

Vibrio cholerae and Vibrio eltor donor strains were obtained using R plasmids RP1ts, pRP19.6, pTH10 and pNS10 which carry IS or Tn elements. Resulting from integration of the plasmids into cholera vibrio chromosome, two types of donors were constructed: those resembling Escherichia coli Hfr's and the strains analogous to F+ or F' donors. The order of some chromosome markers were determined in crosses between the donors obtained and V. cholerae recipient strains.

By electron microscopy and roentgenography glucomannanes from four species of Eremurus have been examined and shown to be polymorphic. Deacetylated glucomannane produces a lamellar form with a high degree of crystallinity. Partially acetylated glucomannane produces a fibrillar form representing a poorly ordered structure with a spaced (4.4 A) position of chains going in the same direction. In plants glucomannane can also exist as fibrils. The factors determining the formation of a certain supramolecular form are acetyl substitutes in the chain of the polysaccharide and the degree of its polymerization. The effect of the supramolecular structure on the properties and biological function of glucomannanes is discussed.

Hemolysin hyper- hypoproductive Vibrio eltor mutants and hemolytically inactive clones were isolated using tetracycline (Tn10) and chloramphenicol (Tn9) resistance transposons. Chromosomal localization was demonstrated for hml gene(s) involved in hemolysin production. Mutations responsible for an altered hemolysin production are likely to be situated closely to pur ura arg met chromosomal markers.

The specificity of integration of chloramphenicol resistance transposon (Tn9) into Vibrio El Tor chromosome was studied. As shown by the isolation of auxotrophs offerring various nitritional requirements, Tn9 displayed fairly low specificity in its interaction with the chromosome. When inserted into cholera vibrio chromosome, Tn9 induced secondary rearrangements of genetic material. Nutritional requirements were identified for the polyauxotrophic clones which resulted from the mutations in chromosome region adjacent to the insertion sites of Tn9 and Tn10. Based on these observations a preliminary map was constructed for certain regions of V. eltor chromosome.

Introduction of tetracycline-resistance transposon Tn10 into Rts1 (Km), a plasmid which is temperature-sensitive for DNA replication, made it possible to construct pNS10 (Km Tc) plasmid used as a vehicle for transferring Tn10 from Escherichia coli to cholera vibrio V. El Tor 218 as a result of intergeneric crosses. While being translocated from the plasmid genome to the chromosome of cholera vibrio transconjugants, Tn10 was able of insertion into structural genes thus leading to the formation of auxotrophic mutants. Ability of Tn10 to promote chromosomal rearrangements of the regions linked to Tn-element insertion site was used for identification of some structural genes in the neighbourhood of Tn-element insertion site.

The data of genetic mapping of the cholera toxin regulatory gene by conjugation mating of Vibrio cholerae eltor donor strain with V. cholerae classica recipients are presented. The close genetic linkage of tox locus to pur-63 is shown. The gene order asp - cys - nal - pur-61 - trp - his - pur-63 - tox - ile of the chromosomal region examined is established.

The insertion of transposable deoxyribonucleic acid sequence that specifies chloramphenicol acetyltransferase (Tn9) occurs in the preferential sites of the Escherichia coli K-12 chromosome. It is found that some strains of E. coli K-12 have different preferential sites for the Tn9 insertion, located in various regions of the chromosome. The IS1 element inserted into the galT gene of the N116 strain (insertion mutation galT 116:IS1) serves as a strongly preferential site for the Tn9 insertion in this strain. However, being transferred into the chromosome of the KS836 strain this element loses such preference. It was demonstrated that the transposons (Tn9) inserted into different chromosomal sites are distinguished by such properties as stability, different capability to transposition into genomes of bacteriophages and plasmids, and by the choice of the preferential sites of integration in the course of subsequent insertions.