Cloning and detailed mapping of the fra-ymt region of the Yersinia pestis pFra plasmid]

The genetical libraries of the pFra plasmid of Yersinia pestis genes were obtained by insertion into the PstI, SalGI, EcoRI, XhoI restriction sites of the cosmid vector pHC79. The immunochemical analysis of the recombinant clones has revealed the clones synthesizing the antigen Fl (fraction I) and mouse toxin (Ymt--Yersinia pestis murine toxin). The restriction analysis of the plasmids from antigen synthesizing clones has permitted to construct the detailed physical map of the fra-ymt region of the pFra plasmid the size of 22 kb. The recombinant F1 positive clones of Escherichia coli are able to form at 37 degrees C the capsule-like structure peculiar for Yersinia pestis. The antigen F1 and the mouse toxin were isolated, purified and characterized. The antigen F1 is an 1-2 Md polymer containing a 16 kDa protein subunit. The mouse toxin a 240 kDa protein consisting of 61 kDa subunits. The nucleotide sequence of ymt gene has been defined.     

The effect of the bacterial genome on the expression and behavior of the plasmid determining the Ca2+-dependence of the plague pathogen]

The conjugative cointegrate containing the 47 Md plasmid of Yersinia pestis has been transferred into the strains of the different Yersinia (Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica) and Escherichia coli CA. There appeared in the populations of recombinant Yersinia under the conditions of Ca2+ deficit at 37 degrees C the cells coming into the stasis stage or dying. It was shown on the model of Yersinia enterocolitica that bacterial lethality might be prevented by exclusion of the sheep blood from Ca2+ deficient medium. Ca(2+)-dependence was not expressed in Escherichia coli cells in which the cointegrates were prone to deletions although the cad-genes were preserved intact. The latter conclusion is based on the positive reciprocal transfer of the Cad(+)-marker into Yersinia pestis cells.     

Study of protein and carbohydrate products, synthesized by Yersinia pestis under conditions imitating the internal environment of mammalian phagolysosomal macrophages

Acid shift (pH 4.0) of liquid nutrient medium containing 20 mM Mg2+ created conditions in vitro simulating the internal environment of phagolysosome into which Yersinia pestis captured by a macrophage get in vivo. The capacity of Y. pestis to survive and multiply under these conditions irrespective of the plasmid composition of strains was confirmed experimentally. Y. pestis possesses a specific mechanism of fibrinolytic activity inhibition, preventing proteolytic degradation under the effect of Ca-dependent polypeptide (Yops) fibrinolysin and potentiating, in addition to these latter, the production of the so-called "acid" proteins by Y. pestis, coded for by pCad2+ or chromosome, including the potentially new members of LCR family. The culturing conditions affect the length of O-specific lateral chains of Y. pestis lipopolysaccharide (LPS), which corresponds to LPS SR, but not R form.     

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.     

Expression of Yersinia pestis antigens encoded by the Ca2+-dependence plasmid

Antigens coded by the Ca2(+)-dependance plasmid were found in the cultural medium, cytoplasm and outer membranes of the three monoplasmid (pCadV) strains of Yersinia pestis with the different basic properties. The presence of 20 mM of Mg2+ at least in the medium is necessary for optimal expression of these proteins. The existence of strain differences in the bacterial cells reaction to temperature, cultivation medium has been demonstrated. No difference in the pCad-dependent proteins was found in Yersinia pestis and the causative agents of pseudotuberculosis, enterocolitis.     

Study of the dependence of gene expression of plague pathogen plasmid 65MD on the host bacterium genome

The conjugative cointegrate containing Yersinia pestis 65 Md plasmid coding for the production of traction I antigen and mouse toxin has been transferred into Yersinia pseudotuberculosis, Yersinia enterocolitica and Escherichia coli cells. Analysis of the transconjugants obtained has confirmed the connection of the genetical determinants for the mentioned bacterial products with the 65 Md plasmid. The similar level of fra and tox-genes expression has been found in Yersinia cells while fraction I was not produced in Escherichia coli CA cells. The data on the increased synthesis of fraction I at 40 degrees C as compared with the one at 37 degrees C has been obtained. In some cases the production remained at the same level characteristic of the 37 degrees C. The possibility of formation of different Yersinia Fra+ recombinants in the mixed natural epizootic foci and their role in epizootic infections are discussed.     

Detection and characterization of the plasmids of the plague microbe which determine the synthesis of pesticin I, fraction I antigen and "mouse" toxin exotoxin

Plasmid DNA was isolated from Yersinia pestis strains containing pesticin I or fraction I antigen and "mouse" toxin determinants. Specificity of DNA preparations was studied by using them for transformation of plague agent strains carrying no plasmids. pPstI plasmid (molecular weight 7,0-7,8 MD) encoded pesticin I, fibrinolysin and plasmacoagulase synthesis. Fraction I antigen and "mouse" toxin production determinants were borne on pFraI/Tox plasmid (molecular weight about 50 MD). The observation that some Y. pestis cultures, having lost the ability to synthesize one of pFraI/Tox products, still retained this plasmid in their cells, is regarded as an evidence for a complicated regulation of pFraI/Tox function.     

Virulence-associated plasmids from Yersinia enterocolitica and Yersinia pestis.

A 44-megadalton plasmid associated with virulence and Ca2+ dependence from Yersinia enterocolitica 8081 was compared at the molecular level with a 47-megadalton plasmid associated with Ca2+ dependence from Yersinia pestis EV76. The plasmids were found to share 55% deoxyribonucleic acid sequence homology distributed over approximately 80% of the plasmid genomes. One region in which the plasmids differed was found to contain sequences concerned with essential plasmid functions. Forty-five mutants of Y. pestis were isolated which had spontaneously acquired the ability to grow on calcium-free medium (Ca2+ independence). Of these mutants, 21 were cured of their 47-megadalton plasmid, whereas the remaining had either suffered a major deletion of the plasmid or had a 2.2-kilobase insertion located in one of two adjacent BamHI restriction fragments encompassing approximately 9 kilobases. The inserted sequence was found at numerous sites on the Y. pestis chromosome and on all three plasmids in the strain and may represent a Y. pestis insertion sequence element.     

Isolation of transmissible cointegrates of Yersinia pestis plasmids pYV and pYT with the plasmid RP4::Mu cts 62, IncP1

The transmissible cointegrates of the Yersinia pestis plasmids pYV and pYT with the broad host range plasmid RP4::Mu cts62 of the incompatibility group IncP have been constructed by the in vivo recombination. The cointegrative plasmid pKR14 (pYV76 omega RP4::Mu cts62) conferred on the transconjugants the properties of Ca2(+)-dependence at 37 degrees C, V-antigen synthesis, RP4 plasmid markers (ApR, KmR, TcR), immunity to the lysis by the bacteriophage Mu cts62 and incompatibility with the homologous replicon pYV76. Cointegrates pKR103 and pKR106 (pYT omega RP4::Mu cts62) conferred on the transconjugant clones the ability to synthesize the "mouse" toxin and fraction I. The capability of Escherichia coli cells to synthesize the latter products has been demonstrated together with the deficiency of these cells to transport the synthesized fraction I to the cell surface.     

LcrD, a membrane-bound regulator of the Yersinia pestis low-calcium response.

Yersinia pestis, the etiologic agent of bubonic plague, contains a 75-kb virulence plasmid, called pCD1 in Y. pestis KIM. The low-Ca(2+)-response genes of Y. pestis regulate both bacterial growth and the expression of pCD1-encoded virulence determinants in response to temperature and the presence of Ca2+ or nucleotides. This study characterizes the nucleotide sequence and protein product of the lcrD locus. An lcrD mutant, in contrast to the parent Y. pestis, did not undergo growth restriction or induce strong expression of the V antigen when grown under conditions (37 degrees C, no Ca2+) expected to elicit maximal expression of pCD1 genes. DNA sequence analysis of the cloned lcrD locus showed a single open reading frame that could encode a protein with a molecular weight of 77,804 and a pI of 4.88. LcrD was identified as a 70-kDa inner membrane protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. LcrD membrane topology was investigated by using lcrD-phoA translational fusions generated with the transposon TnphoA. The alkaline phosphatase activities of the resultant hybrid proteins were consistent with a model predicting eight amino-terminal transmembrane segments that anchor a large cytoplasmic carboxyl-terminal domain to the inner membrane.     

Cloning of the locus encoding synthesis of an outer membrane protein (Omp2) of the calcium dependence plasmid of Yersinia pestis (Lehmann, Neumann)

The genetic locus of Yersinia pestis encoding synthesis of a 46-kDa heat-inducible outer membrane protein (Omp2) was cloned into pBR322 plasmid. The Omp2 was shown to be analogous to previously described YopH and Yop2b proteins. The fifth HindIII fragment of 48-MDa calcium dependence plasmid pCad358 mediates production of 31- and 28-kDa proteins, irrespective of orientation of the insertion. A 31-kDa polypeptide seems to correspond to the YopJ described elsewhere. The maps of BamHI and HindIII of pCad358 region studied differed from those described for pCD1 plasmid of Y. pestis KIM. The products encoded by genes from the fragment cloned in the Pgm+ background give rise to considerable growth of Y. pestis within mouse peritoneal macrophages but were not sufficient to cause lethal infectious process.     

Results of screening of plasmids of Yersinia pestis strains from various regions of endemic central-asian plague focus

The results of the plasmid screening of Yersinia pestis strains isolated from four autonomous focuses on the northern border of the Central Asian zone of plague natural focality are presented. The plasmid profile of Yersinia pestis strains from the focuses is characterized as stable and independent of the source and time of strain isolation. The peculiar characteristic of the strains isolated in Tuva is the presence of an "additional" 15-16 Md plasmid in those strains.     

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.     

Consequences of Ca2+ deficiency on macromolecular synthesis and adenylate energy charge in Yersinia pestis.

A 37 but not 26 degrees C virulent Yersinia pestis is known to require at least 2.5 mM Ca2+ for growth; this requirement is potentiated by Mg2+. After shift of log-phase cells (doubling time of 2 h) from 26 to 37 degrees C in Ca2+-deficient medium, shutoff of net ribonucleic acid synthesis preceded that of protein and cell mass. With 2.5 mM Mg2+, about two doublings in cell mass and number occurred before restriction with synthesis of sufficient deoxyribonucleic acid to account for initiation and termination of two postshift rounds of chromosome replication. Temperature shift with 20 mMMg2+ resulted in a single doubling of cell mass and number with one round of chromosome replication. Subsequent to shutoff of ribonucleic acid accumulation, ribonucleoside but not deoxyribonucleoside triphosphate pools became reduced to about 50% of normal values and the adenylate energy change fell from about 0.8, typical of growing cells, to about 0.6. Excretion of significant concentrations of adenine nucleotides under both permissive and restrictive conditions was observed. Only trace levels (less than 0.01 microM ol/g [dry weight]) of guaninosine 5'-diphosphate 3'-diphosphate accumulated under restrictive or permissive conditions; guanosine 5'-triphosphate 3'-diphosphate was not detected. Return of fully restricted cells from 37 to 26 degrees C with Ca2+ resulted in prompt growth, whereas addition of Ca2+ at 37 degrees C was ineffective. This finding indicates that the observed temperature-sensitive lesion in ribonucleic acid synthesis that results in restriction can be prevented but not reversed by cultivation with Ca2+.     

Influence of cultivation conditions on the expression of Yersinia pestis YopE

With the use three types of nutrient media made it possible to study the specific features of the biosynthesis of YopE, one of the main effector proteins, coded by Yersinia pestis virulence plasmid. This protein was proved to be produced practically at all stages of Y. pestis parasitism in the host body. The above-mentioned antigen was found capable of being synthesized, depending on the conditions of Y. pestis cultivation, in the form of membrane-linked (extracellularly and under phagosomal conditions) or secreted substance, mainly in phagolysosome. In the latter case the maximum level of its expression was registered. The experimental confirmation of YopE localization in the form of superficially localized antigen/receptor at the period of the extracellular growth of bacteria is presented, which suggests its important role in the realization of the virulent properties of Y. pestis and, together with the known data on the protective properties of the antigen, indicates the prospects of its use as the basis for the creation of new chemical antiplague vaccine.     

Spectrophotometric determination of photoreceptor cGMP-gated channel Mg2(+)-fluxes using dichlorophosphonazo III.

We have characterised the spectroscopic properties of the metallochromic dye dichlorophosphonazo III and describe its use for the determination of changes of Mg2+ concentration in the micromolar range. Using a previously described reconstitution procedure, we incorporated the cGMP-gated channel from bovine rod photoreceptors into magnesium-containing liposomes and used the dye to monitor cGMP-activated Mg2(+)-efflux. The Km and cooperativity of the cGMP-dependence were identical regardless of whether Mg2+ or Ca2+ was the transported ion, however, the vmax for Ca2+ was more than 2-fold higher than that for Mg2+. We thereby determined a channel selectivity (Ca2+:Mg2+) of 1.0:0.44 in the presence of symmetrical (30 mM) K+. We also describe conditions where Mg2+ or Ca2+ effluxes can be selectively monitored in the presence of each other. This allowed the demonstration that magnesium ions can flow through the cGMP-gated channel even in the presence of an identically directed calcium gradient. Together these results indicate that magnesium ions may enter the photoreceptor rod outer segment cytosol through the cGMP-gated channel under dark conditions, thereby alluding to the existence of an as yet unknown Mg2(+)-extrusion mechanism, distinct from the Na+/Ca2(+)-exchanger, in these cells.     

The use of mini-Mu-replicons for cloning of various structural genes of Yersinia pestis

The possibility to clone the structural leu gene of Yersinia pestis in vivo using the mini-Mu bacteriophages with the inserted plasmid replicones has been demonstrated. The E. coli K12 transductants having obtained the Leu(+)-marker within the cloned 4.8-21 kb fragments stably inherited the leu-gene within the autonomous mini-Mu replicones. The possibility to clone other Yersinia pestis genes by the same technique has been demonstrated.     

Restriction map of pesticinogenicity plasmid pYP1 of Yersinia pestis

The restriction map of Yersinia pestis pesticinogenicity plasmid pYP1 has been constructed with the use of 18 restriction endonucleases. Plasmid dimensions (6.3 Md) have been specified, the genes for pesticin synthesis, for pesticin immunity protein, fibrinolysin and plasmocoagulase have been localized by molecular cloning of single plasmid DNA fragments in vector plasmid pBR322.     

Effect of Ca2+ ions on plasmid transformation of Streptococcus lactis protoplasts

The effects of Mg2+ and Ca2+ ions on the efficiency of the plasmid transformation of lysozyme-treated Streptococcus lactis protoplasts were compared. A 33-megadalton plasmid, pLP712, coding for lactose fermentation and a 6.5-megadalton plasmid, pGB301, coding for erythromycin and chloramphenicol resistance were used as model plasmids, and S. lactis MG1614 was the recipient. Replacing Mg2+ with Ca2+ in the transformation buffer was found to increase transformant frequency more than 10-fold with both plasmids.