Alternative localization of the determinant of pathogenicity coded by the Yersinia pseudotuberculosis pVM82 plasmid]

The chromosomal DNA regions in Yersinia pseudotuberculosis strains occur that are homologous to 25 Md DNA segment of the plasmid pVM82 encoding the bacterial capability of immunosuppression. The character of the chromosomal DNA regions dispersion reacting with the 25 Md segment probes is different in epidemiologically hazardous and nonvirulent strains of Yersinia pseudotuberculosis. The specific DNA regions occur as well as identical ones. The suppression of antibody formation to a number of main Yersinia pseudotuberculosis antigens by epidemiologically hazardous strain is demonstrated. The suppression is analogous to the one previously described for Yersinia pseudotuberculosis strains harbouring the plasmid pVM82.     

A new pathogenic trait encoded by Yersinia pseudotuberculosis pVM82 plasmid

The strains of Yersinia pseudotuberculosis isolated from patients in the course of outbreaks of infection (epidemic strains) were found to possess at least two plasmids with molecular masses of 45 and 82 MD. In contrast, the strains obtained in sporadic cases harbored different sets of plasmids, but never the 82 MD plasmids. These plasmids designated pVM82 and isolated from strains of different geographic regions of the country were identical. pVM82 have no homology with Y. pestis plasmids of the similar size coding for the FraI antigen. The pVM82 DNA was found to be composed of the 57 MD plasmid DNA and the 25 MD fragment of Y. pseudotuberculosis DNA. Using Western blot hybridization technique it was shown that the presence of pVM82 suppressed formation of antibody against some major antigenic determinants of Y. pseudotuberculosis. Immunosuppression took place when the animals were infected with bacteria grown below 20 but not at 37 degrees C. The 57 MD plasmid failed to produce immunosuppression. It was concluded that the 25 MD fragment of pFN82 encoded a novel pathogenic factor responsible for immunosuppression.     

Synthesis of lipopolysaccharides in the bacterium Yersinia pseudotuberculosis: effect of the pVM82 plasmid and growth temperature

The composition and structure of lipopolysaccharides (LPS) of three isogenic strains of Yersinia pseudotuberculosis serovar O:1b (without plasmids (82-) and with plasmids pVM82 (82+) or p57 (57+)) grown at 8 or 37 degrees C were studied by chemical and immunochemical methods, SDS-polyacrylamide gel electrophoresis, and 13C-NMR spectroscopy. At the lower temperature, the (82-) and (82+) strains synthesized S-form of LPS with similar structure characterized by high acylation and immunochemical activity. On the other hand, LPS of the (82+) strain had shorter carbohydrate chains than LPS of the (82-) strain. The contents of LPS were decreased in cells of the plasmid-free strain grown at the higher temperature. LPS isolated from these cells were of the R-form and had low acylation and immunochemical activity. Total LPS content in cells of the (82+) strain did not significantly depend on the growth temperature. LPS of the warm variant of these bacteria contained a polysaccharide fragment and had moderate immunochemical activity. The cells of the (57+) strain at both growth temperatures had low LPS contents and produced LPS of low acylation without O-specific chains (cold variant) or containing O-polysaccharide with low polymerization degree (bacteria grown at 37 degrees C). The data indicate that in the absence of the plasmids, LPS synthesis is encoded by the chromosomal genes in pseudotuberculosis bacteria. Expression of the genes involved in LPS synthesis is regulated by the temperature of bacterial growth. Genes responsible for temperature-dependent regulation of LPS biosynthesis are located on chromosomal DNA. The pVM82 plasmid includes two gene groups; one group is localized in a 57-mD fragment of DNA and inhibits LPS synthesis, suppressing temperature-dependent regulation of the synthesis. The genes located in a 25-mD fragment of the pVM82 plasmid are de-repressors of the 57-mD fragment, and they restore the ability of pseudotuberculosis bacteria to synthesize relatively long LPS at both growth temperatures.     

Structural-functional characteristics of Yersinia pseudotuberculosis plasmid pVM82

The restriction map of Yersinia pseudotuberculosis plasmid pVM82 was established using the "chromosome walking" method. According to transpositional mutagenesis, the plasmid pVM82 appeared to be conjugative and was able to be transmitted from Y. pseudotuberculosis to the E. coli K-12 cells.     

Effects of growth temperature and pVM82 plasmid on fatty acids of lipid A from Yersinia pseudotuberculosis

Effects of cultivation temperature (8 or 37 degrees C) and plasmid profile on the lipid A fatty acids of three isogenic Yersinia pseudotuberculosis strains (plasmidless (82-) and strains containing pVM82 (82+) or p57 (57+) plasmids) obtained by alkaline hydrolysis of the whole bacterial cells and differentiated from fatty acids of other membrane lipids were investigated. On the basis of the analysis, it is concluded that lipids A of all studied samples contain 3-hydroxytetradecanoic and dodecanoic acids, a part of which exists as the 3-dodecanoyloxytetradecanoic derivative. The effect of temperature appears in the higher contents of ester- and amide-linked 3-acyloxyalkanoic residues in lipid A from the "cold" variants of the bacteria and is determined by chromosomal genes. The plasmid effect is seen as various responses of the isogenic derivatives to change of growth temperature: in cells of strains 82+ and 82- grown in the cold, the share of lipid A fatty acids in the total population of cellular fatty acids is reduced, while in strains with plasmid p57 it is increased. The temperature variants of the 57+ strain differ by the low contents of amide-linked 3-acyloxyalkanoic acids. Finally, lack of plasmid pVM82 in the "warm" variants of the bacteria results in accumulation of glycolipid molecules deprived of dodecanoic acid. Correlation between growth temperature and plasmid profiles, on one hand, and lipid A fatty acid composition and potential pathogenic properties of the Y. pseudotuberculosis, on the other hand, and also possible mechanisms of thermal adaptation of this organism are discussed.     

The effect of Yersinia pestis EV76 6 MD plasmid on the composition of outer membrane proteins of Yersinia pseudotuberculosis YPIII]

On the basis of Yersinia pseudotuberculosis strain YPIII the isogenic variants containing the different combinations of 47 Md plasmids from Yersinia pestis or Yersinia pseudotuberculosis cells with the 6 Md pYP plasmid from Yersinia pestis EV (intact or having impaired the pla gene determining the synthesis of plasmocoagulase). The degradation of the secreted proteins encoded by the 47 Md plasmids of Yersinia pestis and Yersinia pseudotuberculosis in the cells harbouring the 6Md pYP plasmid has been registered. Yersinia pseudotuberculosis strain YPIII carrying its own 47Md and pYP plasmids also contained no YOP1 protein, in contract to the parent strain. The damage of the pla gene eliminated the destructive effect on the outer membrane proteins. Imposition of the 47Md and 6Md plasmids from Yersinia pestis in Yersinia pseudotuberculosis cells may be used for obtaining and study of the physiological role of low molecular mass proteins resulting from proteolysis of proteins encoded by the 47Md virulence plasmid of Yersinia.     

Conjugative R-plasmid of facultative methylotrophic bacteria Pseudomonas sp. M

50 Md conjugative plasmid, designated pM3, has been found in the cells from natural isolates of Pseudomonas sp M. The plasmid determines the resistance to tetracycline and streptomycin and is capable of conjugative transfer between the cells of Pseudomonas and Escherichia coli. The conjugative derivatives of pM3 deleted for 14 Md of molecular mass were isolated after acridine dyes treatment of cells harbouring plasmid pM3. The discovered plasmid was not shown to belong to IncP1 incompatibility group.     

Analysis of the plasmid composition of Yersinia pseudotuberculosis strains and its use for typing pseudotuberculosis pathogens

Electrophoresis in agarose gel has been used to study the plasmid spectra of 854 Yersinia pseudotuberculosis strains isolated from different sources. The plasmids found in the microbial strains are represented by the elements with molecular masses 82; 57; 45; 5.5; 4.4; 3.5; 2.7; 2.4; 2.3 Md. The variable spectra of plasmids is peculiar only for serovar I of Yersinia pseudotuberculosis. Plasmids p45 and p82 are classified as the main, while other plasmids as auxiliary ones. In accord with the classification all plasmid containing strains are divided into 8 plasmid strains. Using the proposed method for intraspecific typing of Yersinia pseudotuberculosis permits one to perfect the epidemiological analysis of pseudotuberculosis infection and make concrete the direction of prophylactic and antiepidemic measures.     

Characterization of Bacillus stearothermophilus plasmid pab124 and construction of deletion variants

A restriction endonuclease cleavage map of the tetracycline resistance plasmid pAB124, originally isolated from Bacillus stearothermophilus, was constructed using ten enzymes. Tetracycline resistance was associated with a 1 x 95 megadalton (Md) region of pAB124 lying between two EcoRI sites, and this region was circularized to produce a viable tetracycline resistance plasmid (pAB224), with two EcoRI fragments of pAB124 deleted amounting to 0 x 95 Md. A second plasmid (pAB524) with one EcoRI fragment (0 x 6 Md) of pAB124 deleted was also constructed. Restriction endonuclease cleavage maps of pAB224 and pAB524 were constructed.     

Molecular cloning and mapping of a deletion derivative of the plasmid Rts 1

The plasmid pTW20 is a deletion derivative of the kanamycin resistance plasmid Rts1. By digesting pTW20 DNA with EcoRI endonuclease six fragments were generated, and each was cloned in the vector plasmid pACYC184. These cloned EcoRI fragments were further digested with various endonucleases, and the cleavage map of pTW20 was constructed. A SalI fragment (1.5 Md) in E1 (the largest EcoRI fragment; 11.5 Md) contained the genes kan (kanamycin resistance) and puv (uv sensitization of host). An electron microscopy study of a BamHI fragment containing kan revealed the presence of a transposon-like structure in the fragment. The smallest EcoRI fragment E6 (2.0 Md) was capable of autonomous replication in a polA host, indicating that E6 contained replication genes of pTW20. These genes were found to be located on a 1.1-Md HindIII fragment in E6. Two incompatibility genes were identified on the pTW20 genome, one located on each of the fragments E6 and E5 (3.5 Md), and expressed T incompatibility independently. The nature of the temperature sensitivity of pTW20 was discussed.     

Cloning of the replication and incompatibility regions of a plasmid derived from Rts1

A replication region, consisting of a 1.1-megadalton (Md) EcoRI/HindIII fragment, was isolated from an Rts1 derivative plasmid. This 1.1-Md fragment, designated as mini-Rts1, was ligated to either pBR322 or a nonreplicating DNA fragment specifying a drug resistance, and its replication properties were investigated. The mini-Rts1 plasmid was cured at a high frequency at 42 °C, while it was maintained stably at 37 °C despite it existed in low copy number. These behaviors are quite similar to those of Rts1. By dissecting the pBR322:mini-Rts1 chimeric plasmid with AccI endonuclease, an inc region of 0.34 Md in size was cloned, which expressed incompatibility toward Rts1. Proteins encoded on the mini-Rts1 genome were examined in the minicell system, and one specific product of 35,000 daltons in molecular weight was identified. Any polypeptides specific for the 0.34-Md inc⁺ region within mini-Rts1 were not detected.     

Direct repetition of a 1.2 Md DNA sequence is involved in site-specific recombination by the P1 plasmid R68

R68.45, a mutant R68 plasmid, carries a 1.5 Md DNA insertion near its kanamycin-resistance region. This DNA consists of a 1.2 Md DNA repetition of neighbouring R68-DNA and a 0.3 Md “foreign” DNA fragment that is flanked by this direct DNA repeat. This fragment seems to be involved in the formation of R'68.45 plasmids. Duplication of the 1.2 Md DNA sequence is also involved in site-specific recombination events of RP4. This 1.2 Md DNA fragment has the properties of an IS sequence and is denoted IS8.     

Construction of plasmid vectors for gene cloning in Escherichia coli and Bacillus subtilis

The construction and some properties of new hybrid plasmids which are able to replicate in both Escherichia coli and Bacillus subtilis are presented. A 5.5 Md hybrid plasmid pJP9 was constructed from pBR322 (Tc, Ap) and pUB110 (Nm) plasmids. pIM1 (7.0 Md) and pIM3 (7.7 Md) plasmids are its different erythromycin resistant derivatives. Tetracycline, ampicillin, neomycin and possibly erythromycin resistance genes are expressed in E. coli while neomycin and erythromycin resistance genes are expressed in B. subtilis. Insertional inactivation of only one gene is possible using the pJP9 plasmid as a vector in B. subtilis. However, insertional inactivation of at least two different genes can be achieved and monitored in E. coli and B. subtilis transformants in cloning experiments with PIM1 and pIM3 plasmids. Insertional inactivation of antibiotic resistance genes present in pJP9 plasmid was achieved by cloning of Streptococcus sanguis DNA fragments generated by appropriate restriction endonucleases. The pJP9 plasmid and its derivatives were found to be stable in both hosts cells.     

Plasmids of Yersinia pseudotuberculosis

Plasmids with the sizes of 5.7; 51; 70-77; and 120-130 kb were found in six strains among the ten strains collection of Yersinia pseudotuberculosis. The restriction endonucleases analysis. Southern-blot hybridization and physical maps construction were performed for the plasmids. The 70-77 kb plasmids were found to be analogous to the Ca2(+)-dependence plasmid pYVO19 from Yersinia pestis EV76. The difference between the plasmids of this type is in the insertions or deletions located on the similar fragments of the restriction maps. The 51 kb plasmid has no common fragments with the Ca2(+)-dependence plasmids and does not code for virulence properties of the strain harbouring it. No homology is shared by the 5.7 kb plasmid and the 10 kb plasmid from Yersinia pestis EV76. Replicon of the 5.7 kb plasmid has been used to construct the pVS11 vector plasmid.     

Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis

Previously, curing experiments suggested that plasmid pWV05 (17.5 megadaltons [Md]) of Streptococcus cremoris Wg2 specifies proteolytic activity. A restriction enzyme map of pWV05 was constructed, the entire plasmid was subcloned in Escherichia coli with plasmids pBR329 and pACYC184. A 4.3-Md HindIII fragment could not be cloned in an uninterrupted way in E. coli but could be cloned in two parts. Both fragments showed homology with the 9-Md proteinase plasmid of S. cremoris HP. The 4.3-Md HindIII fragment was successfully cloned in Bacillus subtilis on plasmid pGKV2 (3.1 Md). Crossed immunoelectrophoresis of extracts of B. subtilis carrying the recombinant plasmid (pGKV500; 7.4 Md) showed that the fragment specifies two proteins of the proteolytic system of S. cremoris Wg2. PGKV500 was introduced in a proteinase-deficient Streptococcus lactis strain via protoplast transformation. Both proteins were also present in cell-free extracts of S. lactis(pGKV500). In S. lactis, pGKV500 enables the cells to grow normally in milk with rapid acid production, indicating that the 4.3-Md HindIII fragment of plasmid pWV05 specifies the proteolytic activity of S. cremoris Wg2.     

Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis.

Previously, curing experiments suggested that plasmid pWV05 (17.5 megadaltons [Md]) of Streptococcus cremoris Wg2 specifies proteolytic activity. A restriction enzyme map of pWV05 was constructed, the entire plasmid was subcloned in Escherichia coli with plasmids pBR329 and pACYC184. A 4.3-Md HindIII fragment could not be cloned in an uninterrupted way in E. coli but could be cloned in two parts. Both fragments showed homology with the 9-Md proteinase plasmid of S. cremoris HP. The 4.3-Md HindIII fragment was successfully cloned in Bacillus subtilis on plasmid pGKV2 (3.1 Md). Crossed immunoelectrophoresis of extracts of B. subtilis carrying the recombinant plasmid (pGKV500; 7.4 Md) showed that the fragment specifies two proteins of the proteolytic system of S. cremoris Wg2. PGKV500 was introduced in a proteinase-deficient Streptococcus lactis strain via protoplast transformation. Both proteins were also present in cell-free extracts of S. lactis(pGKV500). In S. lactis, pGKV500 enables the cells to grow normally in milk with rapid acid production, indicating that the 4.3-Md HindIII fragment of plasmid pWV05 specifies the proteolytic activity of S. cremoris Wg2.     

Various characteristics of Yersinia pseudotuberculosis determined by plasmid 45 Md associated with calcium dependence

The clones of two types (T+ and T-) have been identified among the strains of Y. pseudotuberculosis. The difference in the morphology of the clones is based on the ability of T+ clones to agglutinate in the process of growth. The identified clones are different in calcium dependence, in the ability to agglutinate and in their virulence for the laboratory animals. The differences have been proved to be connected with the presence of a 45 Md plasmid in T+ cells. Replication of this plasmid is suppressed by the acridine orange dye in concentrations of 12.5 or 25.0 mkg X ml-1. The plasmid is spontaneously lost from the strains during their continuous storage. The microscopy of colonies permits the selection of clones with the parental phenotype from the populations having lost the 45 Md plasmid.     

Connection of outer membrane protein composition in Yersinia pestis cells with intrinsic plasmids]

A set of isogenic derivatives of Yersinia pestis EV strain was obtained including the variants harbouring the different compositions of Yersinia own plasmids. The protein profiles of outer membranes of the set of strains were defined. The polyacrylamide gel electrophoresis has shown the small 6.1 Md plasmid to code an outer membrane protein with mol mass 29 kDa, different from pesticin I, while the heavy 60.0 Md plasmid encodes the 15-16 kDa polypeptide different from monomers of F1 and T-antigens of plague microbe.     

Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid.

A composite plasmid has been constructed in vitro from colicin E1 factor (mass of 4.2 megadaltons [Md]) and nontransmissible resistance factor RSF 1010 (mass, 5.5. Md) deoxyribonucleic acids (DNAs) by the sequential action of Escherichia coli endonuclease (RI (Eco RI) and T4 phage DNA ligase on the covalently closed circular forms of the constituents. The composite plasmid was selected and amplified in vivo by sequential transformation of E. coli C600 with the ligated mixture and selection of transformants in medium containing streptomycin plus colicin E1, followed by amplification in the presence of chloramphenicol and purification of the extracted plasmid by dye-buoyant density gradient centrifugation in ethidium bromide-cesium chloride solution. Treatment of the composite plasmid with Eco RI yielded two fragments with mobilities corresponding to the linear forms of the parental plasmids, whereas Serratia marscesens endonuclease R (SmaR), which introduces a single scission in the colicin E1 factor but not in RSF 1010, convErted the composite plasmid to a single linear molecule (mass, 9.7 Md). Sequential degradation of colicin E1 factor with Sma R and Eco RI produced two fragments with masses of 3.5 and 0.7 Md; sequential degradation of RSF 1010 produced only one fragment (due to the cleavage with Eco RI), and sequential degradation of the composite plasmid produced the expected three fragments--an RSF 1010 Eco RI linear and the two expected products from the colicin E1 factor moiety. The composite plasmid conferred on the host cell resistance to streptomycin, sulfonamides, and colicin E1, but colicin E1 itself was not synthesized. In contrast, colicin E1 was synthesized by cells containing simultaneously both colicin E1 factor and RSF 1010 as separate entities. In the presence of chloramphenicol, the composite plasmid continued to replicate for 6 h. whereas replication of RSF 1010 and chromosomal DNA stopped within 2 h. Continued replication in the presence of chloramphenicol suggests that the replicator of the colicin E1 factor is functional in the composite plasmid.     

A palindromic mutation of the linear killer plasmid k2 of yeast.

Production of the killer toxin in Kluyveromyces lactis is dependent on the presence of two linear DNA plasmids, k1 and k2. We isolated a non-killer mutant, VM5, with a modified plasmid composition. It had lost k1, but conserved k2, and acquired, in addition, three new DNA species. The new species were found to be rearranged derivatives of the k2 plasmid. One of them, pVM5-1, was made of the left terminal 4720 bp sequence of k2, including the inverted terminal repeat, and was organized as a large palindromic dimer molecule. The second, pVM5-2, was made of one strand of the pVM5-1 palindrome, folded into a hairpin structure. Like normal k2, pVM5-1 and 2 were present in a high copy number. The third species, pVM5-x, of variable size, was also a deletion product of k2, but not palindromic, and did not contain the terminal repeat. Genetic analysis showed that the presence of the palindromic derivatives appeared to destabilize the normal k2 genome, leading to gradual accumulation of plasmid-less cells.