Phosphatidylcholine-specific phospholipase C and sphingomyelinase activities in bacteria of the Bacillus cereus group.

Bacillus anthracis is nonhemolytic, even though it is closely related to the highly hemolytic Bacillus cereus. Hemolysis by B. cereus results largely from the action of phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelinase (SPH), encoded by the plc and sph genes, respectively. In B. cereus, these genes are organized in an operon regulated by the global regulator PlcR. B. anthracis contains a highly similar cereolysin operon, but it is transcriptionally silent because the B. anthracis PlcR is truncated at the C terminus. Here we report the cloning, expression, purification, and enzymatic characterization of PC-PLC and SPH from B. cereus and B. anthracis. We also investigated the effects of expressing PlcR on the expression of plc and sph. In B. cereus, PlcR was found to be a positive regulator of plc but a negative regulator of sph. Replacement of the B. cereus plcR gene by its truncated orthologue from B. anthracis eliminated the activities of both PC-PLC and SPH, whereas introduction into B. anthracis of the B. cereus plcR gene with its own promoter did not activate cereolysin expression. Hemolytic activity was detected in B. anthracis strains containing the B. cereus plcR gene on a multicopy plasmid under control of the strong B. anthracis protective antigen gene promoter or in a strain carrying a multicopy plasmid containing the entire B. cereus plc-sph operon. Slight hemolysis and PC-PLC activation were found when PlcR-producing B. anthracis strains were grown under anaerobic-plus-CO(2) or especially under aerobic-plus-CO(2) conditions. Unmodified parental B. anthracis strains did not demonstrate obvious hemolysis under the same conditions.

Genetic organization of the Francisella plasmid pFNL10.

We report here the molecular characterization of pFNL10, a 3990-bp cryptic plasmid of Francisella novicida-like F6168. The plasmid was maintained in F. novicida Utah 112 and F. tularensis LVS strains. We sequenced the entire plasmid and found six open reading frames (ORFs)-ORF1, ORF2, ORF3, ORF4, ORF5, and ORFm. ORF3, ORF4, ORF5, and ORFm are located on the same strand, and we designated it the plus strand. ORF1 and ORF2 are on the complementary strand. The ORFs appear to be arranged in two operons, one comprising ORF5 and ORF4 and the other ORF1 and ORF2. There exist two distinct promoters similar to the Escherichia coli sigma(70) promoter, one 5' to ORF1-ORF2 operon and the other 5' to ORF5-ORF4 operon. We found that in both promoters the transcriptional start is an adenosine. ORF3 is positioned in tandem with ORF5-ORF4, but has its own transcriptional start, a thymidine. However, sequence analysis revealed no recognizable promoter in physical proximity to ORF3. Sequence analysis revealed transcriptional terminators immediately downstream of the two operons. Experimental results showed that the ORF1-ORF2 terminator is authentic. But we could not definitively confirm the ORF5-ORF4 terminator. Two sets of direct repeats, one 31 and the other 13 bp, characteristic of ori are positioned between the two promoters. ORF1 encodes a protein that bears homology to the replication initiation protein RepA of various bacteria, and disruption of this ORF indeed blocked pFNL10 replication. In contrast, ORF2 disruption caused formation of plasmid multimers, suggesting aberrant replication. Our analysis also suggests that pFNL10 replicates by the theta mode. The ORF5-ORF4 operon resembles the phd-doc operon of Escherichia coli bacteriophage P1, but the significance of this similarity is unclear.

Nucleotide sequence, structural organization, and functional characterization of the small recombinant plasmid pOM1 that is specific for Francisella tularensis.

pOM1 is a recombinant 4442-bp plasmid that includes the replicon of the Francisella novicida-like strain F6168 cryptic plasmid pFNL10 and the tetracycline resistance gene (tetC) of plasmid pBR328. pOM1 can stably replicate and is maintained in Francisella tularensis biovars tularensis, palaearctica, and palaearctica var. japonica. The replicon of pOM1 includes the ori region and the repA gene. The ori region, located upstream of the repA gene includes two sets of 31- and 13-bp direct repeats (DR), with AT-rich regions preceding each of the DRs. Two putative promoters of the repA gene were found connected with the DR regions. A 40-kDa protein was encoded by the repA gene and found essential for replication. Expression of the tetC gene is regulated by an Escherichia coli sigma(70)-like promoter and is dependent on the F. tularensis strain and its environment.

[Monitoring of life-threatening infection pathogens in relation to the problem of prediction of critical situations]. / Monitoring vozbuditelei opasnykh infektsii v sviazi s problemoi prognozirovaniia kriticheskikh situatsii.

The epidemic situation in the context of many infectious diseases caused by bacteria is presently assessed as being poor in Russia and other countries. The spectrum of the pathogens that can deteriorate epidemic well-being is highly wide. The epidemic situation in terms of many infectious diseases, including those caused by such causative agents as Bacillus anthracis, Vibrio cholerae, Yersinia pestis, Francisella tularensis and others may deteriorate due to the emergence of their modified forms owing to their specific variability. The above generates the necessity of improving controlling measures or developing the techniques for monitoring the pathogens of infectious diseases, including those in the framework of international cooperation.

[pCSE4 plasmid for cloning of promoter-containing DNA fragments in francisella tularensis]. / Plazmida pCSE4 dlia klonirovaniia promotorsoderzhashchikh fragmentov DNK v francisella tularensis.

Recombinant pCSE4 plasmid has been constructed, which contains the cat gene of the Tn9 without its own promoter and with the restriction BamH1 site in front of the Shine-Delgarno region of this gene. pCSE4 is consistently inherited in the cells of F. tularensis and E. coli and makes the cells of both microorganisms to be resistant to tetracycline. By cloning the Sau3A fragments of F. tularensis chromosomal DNA at the BamH1 site of pCSE4. Promoter-containing DNA fragments were selected. The plasmids with such chromosomal DNA fragments retained their structural integrity and functional activity in F. tularensis.

Expression of cereolysine AB genes in Bacillus anthracis vaccine strain ensures protection against experimental hemolytic anthrax infection.

The cereolysin AB genes from Bacillus cereus VKM-B164 have been expressed in Bacillus anthracis strains: virulent H-7 (PXO1, PXO2), vaccine STI-1 (PXO1), 221 (without its own plasmids). Expression was achieved by cloning the genes in a high copy number plasmid pE194. This construct was integrated with host genomes in amplified form. Gold hamsters were vaccinated with parental and recombinant B. anthracis STI-1 and 221 strains and challenged with virulent ones subcutaneously. Gold hamsters vaccinated with 221 strains showed, absence of protection. STI-1 immunisation protected against the H-7 strain, but did not protect against the recombinant strain. STI-1 recombinant strain protected gold hamsters against the H-7 as well as the recombinant H-7 strains. The results describe the modulation of immunopathogenic properties of B. anthracis due to expression of cereolysin AB genes.

[Erysipelothrix rhusiopathiae: plasmids, resistance to antibacterial drugs]. / Erysipelothrix rhusiopathiae: Plasmidy, ustoichivost' k antibakterial'nym preparatam.

The plasmid profile, virulence and antibacterial drug susceptibility of various strains of E. rhusiopathiae were determined. No correlations between the virulence of the strains, their antibiotic resistance and the plasmid content were detected. Structural and functional analysis of one of the isolated plasmids was carried out to use the plasmid as a vector in the genetic study of E. rhusiopathiae.

[Behavior of heterologous recombinant plasmid pCET in cells of Bacillus anthracis]. / Povedenie geterologichnoi rekombinantnoi plasmidy pCET v kletkakh Bacillus anthracis.

Recombinant plasmid pCET was constructed in vivo in cells of enteric and hay bacillus, on the basis of plasmids pC194, and pBC16. Plasmid pCET inherits marker genes of antibiotic resistance from parental plasmids. Anthrax cells were transformed by the recombinant plasmid developed. The behavior of this plasmid was studied in vegetative Bacillus anthracis cells, which did not pass through the sporulation stage and were cultivated at temperatures permissive for the replicon of plasmid pE194. Under these conditions, plasmid pCET was shown to replicate autonomously, regardless of the host chromosome, and to retain its structure, irrespective of the recipient strain. In this case, the phenotype of transformants fully corresponded to the genotype of plasmids inherited. Elevation of the cultivation temperature of strains Bac, anthracis (pCET) up to 44 degrees C led to the elimination of plasmid pCET from cells of anthrax microbe under conditions nonselective for plasmid pCET and its integration with the host chromosome under selective conditions. The frequency of plasmid pCET integration into the chromosome was approximately 10(-1) for all Bac. anthracis strains studied. In population of vegetative cells of strains Bac. anthracis (pCET), which passed through the sporulation stage under selective for plasmid pCET conditions, DNA of plasmid pCET was detected only in the state integrated with the chromosome. Irrespective of the reasons leading to the integration of plasmid pCET into the Bac. anthracis chromosome, all strains inheriting this DNA within their own genome lost the resistance to tetracycline observed in strains with the extrachromosomal plasmid location. Genome amplification of plasmid pCET in the chromosome of Bac. anthracis was detected.

Development of novel vaccines against anthrax in man.

It has been shown that antianthrax immunity induced by the novel vaccine proposed has not only antitoxic, but also antispore character. The whole complex of antigens, namely surface spore antigens, surface antigens of cell wall and toxin components is required for the induction of strong and stable immunity against anthrax. The STI-1 vaccine strain with introduced resistance to several antibiotics seems to be promising for prophylaxis and treatment of anthrax in case of emergency, especially if antibiotic pretreatment could be expected. The technology for submerged cultivation of Bacillus anthracis vaccine strain and for the development of an anthrax vaccine to be used in human medicine is proposed on the basis of the conception of the immunogenesis.

[The additive synthesis of a regulatory peptide in vivo: the administration of a vaccinal Francisella tularensis strain that produces beta-endorphin]. / Additivnyi sintez reguliatornogo peptida in vivo: vvedenie vaktsinnogo shtamma Francisella tularensis, produtsiruiushchego beta-éndorfina.

It has been shown, that vaccine strain of tularemia microbe, F.T.ISE., which produced recombinant beta-endorphin, when administered to CBA mice. It was shown to increase in the threshold level of pain sensitivity and is associated with peptides associated changes the stereotypic behavior. The observed correlated in time with the pattern of the dynamics of culture in the experimental animals and were associated with the level of recombinant beta-endorphin synthesis.

[Characterization of a Rif-R population of Bacillus anthracis]. / Izuchenie svoistv Rif-R-populiatsii sibireiazvennogo mikroba.

Formation of spontaneous RifR mutants was detected in the populations of various strains of Bacillus anthracis (STI-1, Sterne and CH-7) at a rate of 10(-8) per 1 CFU. The levels of the rifampicin resistance in the mutants were different, the MIC ranged from 16 to 512 micrograms/ml. The clones of the RifR population of the virulent strain CH-7 were heterogeneous in the morphological properties of the colonies and cells, the capacity for the synthesis of the toxin and capsule, the sporulation and virulence. The heterogeneity did not correlate with the levels of the antibiotic resistance. Among the clones of the RifR population there were detected deletion variants by the capacity for the synthesis of the toxin and capsule along with the complete ones. The rifampicin therapy of the infection caused by the complete clone was not efficient. The RifR mutation in B. anthracis did not result in cross resistance to penicillins, cephalosporins, tetracyclines, aminoglycosides, macrolides and chloramphenicol.

[Interaction of bacillus anthracis with benzylpenicillin in vivo and in vitro]. / Issledovanie vzaimodeistviia sibireiazvennogo mikroba s benzilpenitsillinom v usloviiakh in vivo i in vitro.

Interaction of the cells of Bacillus anthracis strain CH-7 with benzylpenicillin was studied. The cells of strain CH-7 were shown to contain the penicillinase gene in the repressed state. Spontaneous derepression of the gene at a rate of 10(-8) resulting in the synthesis of penicillinase was observed. Penicillinase was synthesized constitutionally and its synthesis did not depend on the presence of benzylpenicillin in the cultivation medium. The therapeutic effect of benzylpenicillin in the treatment of the experimental infection induced by the B. anthracis strain producing penicillinase was estimated. The efficacy was shown to depend on the time of the beginning of the antibiotic therapy. When the clinical signs of the infection were evident in the animals contaminated with the penicillinase-producing strain of B. anthracis, their treatment with the mean daily doses of benzylpenicillin failed.

[Comparison of therapeutic effects of antibiotics of the tetracycline group in the treatment of anthrax caused by a strain inheriting tet-gene of plasmid pBC16]. / Sravnenie terapevticheskogo éffekta antibiotikov tetratsiklinovoi gruppy sibirskoi iazvy, vyzyvaemoi shtammom, nasleduiushchim tet-gen plazmidy pBC16.

In vivo and in vitro efficacy of tetracyclines was studied with respect to anthracic infection induced by a tetracycline-resistant resistant strain containing plasmid pBC16. The plasmid-containing strain was resistant to tetracycline, doxycycline and minocycline, the MICs exceeding those for the initial strain 500, 640 and 80 times, respectively. There was no therapeutic effect of tetracycline and doxycycline in the treatment and urgent prophylaxis of anthracic infection caused by the tetracycline-resistant strain of Bacillus anthracis. High therapeutic efficacy of minocycline in the average therapeutic concentrations was shown irrespective of the contaminating doses and strains. Minocycline was recommended for treatment and urgent prophylaxis of anthracic infection caused by tetracycline-resistant B. anthracis strains.

[Behavior of Sa plasmid in tularemia pathogen cells]. / Povedenie plazmidy Sa v kletkakh tuliaremiinogo mikroba.

The genome of Sa plasmid is shown to be a subject of genetical rearrangements in Francisella tularensis cells. The rearrangements either result in plasmid integration into the host cell genome or intramolecular amplification of cat-gene with the subsequent excision and recombination of the derivative plasmids. Stable inheritance of the plasmid is registered after integration while plasmid elimination occurs in case of extrachromosomal localisation.

[Study of CAT gene expression in Sa and pC194 plasmids in Escherichia coli, Francisella tularensis, and Bacillus subtilis cells]. / Issledovanie ékspressii CAT-genov plazmid Sa i pC194 v kletkakh Escherichia coli, Francisella tularensis i Bacillus subtilis.

The unit activities were defined for chloramphenicol-acetyltransferases coded for by the cat-genes of the plasmids Sa and pC194 in Francisella tularensis, Escherichia coli and Bacillus subtilis cells. Francisella tularensis cells were shown to hold intermediate position between Escherichia coli and Bacillus subtilis cells in their ability to express the genes of the different taxonomic origin. The direct dependence was found between the dose of the gene coding for chloramphenicol-acetyltransferase synthesis and efficiency of the gene expression, minimal inhibiting concentration of the antibiotic and colony size on the media containing chloramphenicol.

[Transfer of bacteriophage PRD1 genes into modified plasmid Sa of Escherichia coli and Francisella tularensis cells]. / Perenos genov bakteriofagov PRD1 v modifitsirovannye plazmidoi Sa kletki Escherichia coli i Francisella tularensis.

The donor specific bacteriophage PRDI has been shown to mediate the genes transfer into Escherichia coli and Francisella tularensis cell under certain conditions. It is necessary for the process that the recipient cells inherit the plasmids determining absorbtion of bacteriophages on the cellular surface while the transferred genes are able to be expressed. The frequencies of the tet-gene transfer from the plasmid pSKFT5 into Escherichia coli and Francisella tularensis 15 cells inheriting the plasmid Sa are, correspondingly, 10(-6) and 10(-7) clones per bacteriophage plaque.

[Features of the interaction of Escherichia coli and Francisella tularensis RNA polymerases with hybrid plasmids bearing fragments of Francisella tularensis chromosomal DNA]. / Osobennosti vzaimodeistviia RNK-polimeraz Escherichia coli i Francisella tularensis s gibridnymi plazmidami, nesushchimi fragmenty khromosomnoi DNK Francisella tularensis.

Hybrid plasmids containing the fragments of Francisella tularensis chromosomal DNA and capable of tet-gene expression both in Escherichia coli and Francisella tularensis cells were constructed. The regions of francisella chromosomal DNA binding the RNA-polymerases of Escherichia coli and Francisella tularensis were found by the electron microscopy technique. Interconnection of those regions with the expression of tet-gene of the hybrid plasmids was demonstrated.

[Comparison of the structural-functional properties of DNA-dependent RNA polymerase of the tularemia microbe and intestinal bacterial]. / Sravnenie strukturno-funktsional'nykh svoistv DNK-zaviskmykh RNA- polymeraz tuliaremiinogo mikroba i kishechnoi palochki.

The subunit compositions of RNA-polymerases from Escherichia coli and Francisella tularensis were compared. The activities of the enzymes on the corresponding chromosomal DNAs and their mixtures were defined.