Influence of chemical analogue of extracellular microbial autoregulators on antilysozyme activity of bacteria

Modifying action of C7-alkyloxybenzol (methylresorcin) on the antilysozyme activity (ALA) of opportunistic microorganisms (Bacillus cereus, Klebsiella pneumoniae, and Escherichia coli) was studied. C7-alkyloxybenzol (C7-AOB, methylrezorcin), which was used as chemical analogue of microbial autoregulators, was added to growth medium containing microorganisms, which were cultivated until entered stationary phase. Isolation of clones was performed by seeding of 24-hours broth culture on solid growth medium, and then ALA was measured using photometric method. Modifying action of C7-AOB on ALA characteristic-based population structure of B.cereus, K. pneumoniae, and E. coli was revealed. Maximal effect was detected when the concentration of C7-AOB was in range 1-10 mcg/ml. Decrease of mean ALA level caused by C7-AOB was linked to decrease of proportion of clones with high and intermediate ALA level, increase of proportion of clones with low level of lysozyme inhibitor, and emergence of clones lacking ALA in the population.     

Plasmid transformation of Bacillus cereus protoplasts

The process of polyethyleneglycol-induced plasmid transformation of Bacillus cereus protoplasts was studied. Plasmid transfer into Bacillus cereus strains was demonstrated with the frequencies 1.3.10(1)-1.6.10(2) transformants per 1 mkg of plasmid DNA. The plasmids transferred are stably inherited by Bacillus cereus cells causing tetracycline resistance (pBC16) or kanamycin resistance (pUB110 and pBD64). The proposed method can be used for construction of Bacillus cereus strains having the plasmid determined characteristics.     

Investigation of ampicillin effect on morphological and mechanical properties of Escherichia coli and Bacillus cereus cells with atomic force microscopy

The effect of subbacteriostatic concentrations of ampicillin on morphological and mechanical properties of gramnegative and grampositive cells of Escherichia coli K12 TG1 and Bacillus cereus IP 5832 respectively was studied with atomic force microscopy. Significant heterogeneity of the bacterial populations was shown by the character of the response to the antibiotic effect. The common feature was increase of the cell size likely due to the effect of the inner osmotic pressure on the lowered cell wall strength. In the E. coli population there were besides observed anomalous elongated cells with signs of septation disorder, as well as their structurs, lacking the cytoplasmic liquid fraction. In the B. cereus the inner osmotic pressure mainly enlarged the cell cross section, changing the cell shape from rod to sphere, that was accompanied by significant impairment of the surface structure with liberation of the peptidoglycane fragments to the medium. The particular features of the E. coli K12 TG1 and B. cereus IP 5832 respond to the ampicillin effect were attributed to the differences in the structure of their cell wall, also due to specific properties of the peptidoglycane synthesis and three-dimensional organization.     

Corrected Version Distribution of Heterogeneous and Homologous Plasmids in Bacillus spp

A total of 75 strains (including 5 reference strains) of Bacillus amyloliquefaciens, B. cereus, B. circulans, B. licheniformis, B. megaterium, B. pumilus, B. sphaericus, B. subtilis, and B. thuringiensis and 36 species-unidentified Bacillus strains were surveyed for plasmids by cesium chloride-ethidium bromide equilibrium centrifugation of cell lysates in a study of antibiotic resistance in host cells. Of the 111 strains, 13 (including 3 reference strains) were found to harbor plasmids, and 5 of the 13 showed antibiotic resistance. This antibiotic resistance appeared not to be due to the plasmids, however, because the trait was not cured by cultivation of cells in nutrient medium containing ethidium bromide (1 mug/ml), sodium dodecyl sulfate (0.2 mug/ml), or novobiocin (1 mug/ml), except in one strain, in which kanamycin and streptomycin resistances were cured by novobiocin. One strain of B. amyloliquefaciens, S294, was found to harbor a plasmid, pFTB14, which differed from the plasmid species of classes 1 to 6 in B. subtilis and B. amyloliquefaciens, as determined by restriction analysis and DNA contour length determination. However, in DNA-DNA hybridization on a filter after Southern blotting from an agarose gel, the pFTB14 DNA hybridized with plasmids of classes 1 to 5. Three strains of B. thuringiensis each carried at least 4 to 11 plasmid species, whereas no plasmids were detected in four strains of B. cereus, which, in relation to B. thuringiensis, is closely related taxonomically and has highly homologous DNA sequences. The plasmid DNAs prepared from species other than B. subtilis and B. amyloliquefaciens did not hybridize with that of pFTB14.     

Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis.

A number of plasmids have been isolated as covalently closed circular DNAs from strains of Bacillus cereus and B. subtilis. From 12 out of 15 strains of B. cereus, plasmids could be isolated. Most of the B. cereus strains contained two or more plasmids. Their molecular weights ranged from 1.6 X 10(6) to 105 X 10(6). Bacteriocin production could be attributed to a 45 X 10(6)-dalton plasmid (pBC7) from B. cereus DSM 336, and tetracycline resistance to a 2.8 X 10(6) plasmid (pBC16) from B. cereus GP7. Two streptomycin-resistant strains of B. subtilis harbored plasmids of 5.2 X 10(6) and 9 X 10(6), respectively, which were, however, not correlated with the antibiotic resistance. The plasmid carrying resistance to tetracycline, pBC16, which was originally isolated from B. cereus, could be subsequently transformed in B. subtilis, where it is stably maintained.     

A RAPD-PCR method for the rapid detection of Bacillus cereus

Distinction of Bacillus cereus from other closely related bacilli is challenging and new efficient methods are continually demanded. From our previous work on RAPD profiles of bacilli, we found a possibility that B. cereus strains could be distinguished from other bacilli. In this work, RAPD-PCR profiles of B. cereus strains were obtained using a 10-mer (S30) as a primer, and a B. cereus specific 0.91-kb band was produced from all tested strains. The RAPD-PCR procedure also successfully detected B. cereus from spiked cheonggukjang when B. cereus cells were present at more than 10(2)/g sample.     

The antilysozyme activity of Mycobacterium tuberculosis L forms

The method for the detection of antilysozyme activity (ALA) in M. tuberculosis L forms was developed. The level of ALA in M. tuberculosis L forms isolated from patients with different clinical forms of the disease varied within 1-5 micrograms. M. tuberculosis L forms with the ALA level > 4 micrograms were isolated from patients with the progressing course of the disease. The method for the prognostication of the course of the tuberculous process in the lungs by the results of the antilysozyme test was proposed.     

Cloning and expression in Escherichia coli and Bacillus subtilis of the hemolysin (cereolysin) determinant from Bacillus cereus.

From a cosmid gene bank of Bacillus cereus GP4 in Escherichia coli we isolated clones which, after several days of incubation, formed hemolysis zones on erythrocyte agar plates. These clones contained recombinant cosmids with B. cereus DNA insertions of varying lengths which shared some common restriction fragments. The smallest insertion was recloned as a PstI fragment into pJKK3-1, a shuttle vector which replicates in Bacillus subtilis and E. coli. When this recombinant plasmid (pJKK3-1 hly-1) was transformed into E. coli, it caused hemolysis on erythrocyte agar plates, but in liquid assays no external or internal hemolytic activity could be detected with the E. coli transformants. B. subtilis carrying the same plasmid exhibited hemolytic activity at levels comparable to those of the B. cereus donor strain. The hemolysin produced in B. subtilis seemed to be indistinguishable from cereolysin in its sensitivity to cholesterol, activation by dithiothreitol, and inactivation by antibodies raised against cereolysin. When the recombinant DNA carrying the cereolysin gene was used as a probe in hybridization experiments with chromosomal DNA from a streptolysin O-producing strain of Streptococcus pyogenes or from listeriolysin-producing strains of Listeria monocytogenes, no positive hybridization signals were obtained. These data suggest that the genes for these three SH-activated cytolysins do not have extended sequence homology.     

Insights into the genetic organization of the Bacillus mycoides cryptic plasmids pDx14.2 and pSin9.7 deduced from their complete nucleotide sequence

Bacillus mycoides, a member of the Bacillus cereus group of bacteria, can be easily distinguished from close species because of colony shape, made by filaments of cells, resembling fungal hyphae, curving clock- or counterclockwise depending on the strain. Two plasmids, one from a strain curving to the right (pDx14.2), the other from a strain curving to the left (pSin9.7), were sequenced and analyzed for gene content and replication mode. Rolling-circle replication modules and mobilization proteins were found, very similar to those of other plasmids of the B. cereus group bacilli, mostly Bacillus thuringiensis living in the same ecosystem, suggesting active plasmid exchange in nature.     

Interspecies transduction of plasmids among Bacillus anthracis, B. cereus, and B. thuringiensis

Bacteriophage CP-51, a generalized transducing phage for Bacillus anthracis, B. cereus, and B. thuringiensis, mediates transduction of plasmid DNA. B. cereus GP7 harbors the 2.8-megadalton multicopy tetracycline resistance plasmid, pBC16. B. thuringiensis 4D11A carries pC194, the 1.8-megadalton multicopy chloramphenicol resistance plasmid. When phage CP-51 was propagated on these strains, it transferred the plasmid-encoded antibiotic resistances to the nonvirulent Weybridge (Sterne) strain of B. anthracis, to B. cereus 569, and to strains of several B. thuringiensis subspecies. The frequency of transfer was as high as 10(-5) transductants per PFU. Tetracycline-resistant and chloramphenicol-resistant transductants contained newly acquired plasmid DNA having the same molecular weight as that contained in the donor strain. Antibiotic-resistant transductants derived from any of the three species were effective donors of plasmids to recipients from all three species.     

The isolation of strains of Bacillus subtilis showing improved plasmid stability characteristics by means of selective chemostat culture

A pUB110-derived plasmid encoding chloramphenicol resistance, kanamycin resistance and high-temperature alpha-amylase showed a high degree of segregational instability when inserted into Bacillus subtilis. In an attempt to obtain stable derivatives, the organism was grown in chemostat culture in the presence of chlorampheniol. It was periodically found necessary to increase the concentration of chloramphenicol in the medium feed in order to avoid plasmid loss. Strains were isolated after 19 and 160 generations, which showed high levels of plasmid stability. This characteristic appeared to be genotypic. No detectable difference in plasmid copy number was found between the original and the improved strains. The stability characteristics resided in the host, rather than in the plasmid. Stable isolates possessed elevated MICs for both chloramphenicol and kanamycin. Their maximum specific growth rates were higher than that of the original strain, and similar to that of the plasmid-free parent strain.     

The region controlling the thermosensitive effect of plasmid Rts1 on host growth is separate from the Rts1 replication region.

Rts1 is a high-molecular-weight (126 x 10(6)) plasmid encoding resistance to kanamycin. It expresses unusual temperature-sensitive phenotypes, which affect plasmid maintenance and replication, as well as host cell growth. We have cloned the essential replication region of Rts1 from pAK8, a smaller derivative which is phenotypically similar to Rts1. Restriction endonuclease digests of isolated pAK8 deoxyribonucleic acid were allowed to "self-ligate" (ligation without an additional cloning vector) and subsequently were used to transform Escherichia coli strain 20SO to kanamycin resistance. Screening of these strains for the phenotypes of thermosensitive host growth and temperature-dependent plasmid elimination demonstrated that these two properties were expressed independently. Furthermore, it was shown that the Rts1 replication locus per se is not necessarily responsible for altered host growth at the nonpermissive temperature. The kanamycin resistance fragment of pAK8 was also cloned into pBR322. Electrophoretic analysis of BamHI restriction enzyme digests of this plasmid and similar digests of an Rts1 miniplasmid has allowed the identification of an 18.6-megadalton fragment carrying the replication locus and a 14.1-megadalton fragment carrying the kanamycin resistance gene.     

Antilysozyme activity and antibiotic resistance of periulcerous zone microflora in patients with gastric and duodenal ulcer

Antilysozyme activity (ALA) as well as antibiotic resistance were detected in 133 microbial cultures isolated from bioptic specimens of the mucous membrane of the ulcerous and periulcerous zones, taken from patients with gastric and duodenal ulcer. In 85.7-94.7% of cases Gram positive cocci and in 62.5% of cases Gram-positive bacilli showed no ALA. 50% of Gram negative bacteria cultures lacked ALA, while the remaining 50% exhibited this activity, on the average, 2.36 +/- 1.40 mkg/ml. Lysozyme activity was determined in 33.3% of the isolated staphylococci strains both with and without ALA. Staphylococci isolated from the gastric mucosa of healthy controls had no ALA in 33.3% of cases, and in 66.7% of cases ALA was equal to 2 mkg/ml. Gram positive coccal microflora showed, mainly, high sensitivity to antibiotics. In Gram negative bacteria antibiotic resistance was determined in 44.3 +/- 21.2% of the isolates. In Gram negative microorganisms correlation between ALA and antibiotic resistance was observed. From the periulcerous zone of patients with gastric and duodenal ulcer persistence associated Gram negative microorganisms were mainly isolated.     

Self-transmissible plasmid in Zymomonas mobilis carrying antibiotic resistance.

The cryptic plasmid pRUT41 from Zymomonas mobilis was examined for its biological properties. This plasmid was found to be conjugally transferred from Z. mobilis CP4 to Escherichia coli BM21 and to carry genes for antibiotic resistance (gentamicin, kanamycin, and streptomycin). Covalently closed circular plasmid DNA was isolated from eight transconjugants of E. coli BM21. These plasmids were identical in mobility on agarose gels and exhibited the same restriction patterns as the native pRUT41 plasmid isolated from Z. mobilis. The plasmid location of the antibiotic resistance genes was further confirmed by transforming E. coli BM21 with isolated pRUT41 plasmid from strain CP4 and with plasmids from the transconjugants of BM21. Resistance to streptomycin, kanamycin, and gentamicin was tightly linked and transferred together in all cases.     

Self-transmissible plasmid in Zymomonas mobilis carrying antibiotic resistance

The cryptic plasmid pRUT41 from Zymomonas mobilis was examined for its biological properties. This plasmid was found to be conjugally transferred from Z. mobilis CP4 to Escherichia coli BM21 and to carry genes for antibiotic resistance (gentamicin, kanamycin, and streptomycin). Covalently closed circular plasmid DNA was isolated from eight transconjugants of E. coli BM21. These plasmids were identical in mobility on agarose gels and exhibited the same restriction patterns as the native pRUT41 plasmid isolated from Z. mobilis. The plasmid location of the antibiotic resistance genes was further confirmed by transforming E. coli BM21 with isolated pRUT41 plasmid from strain CP4 and with plasmids from the transconjugants of BM21. Resistance to streptomycin, kanamycin, and gentamicin was tightly linked and transferred together in all cases.     

Identification and partial characterization of the nonribosomal peptide synthetase gene responsible for cereulide production in emetic Bacillus cereus

Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Due to its chemical structure, (D-O-Leu-D-Ala-L-O-Val-L-Val)(3), cereulide might be synthesized nonribosomally. Therefore, degenerate PCR primers targeted to conserved sequence motifs of known nonribosomal peptide synthetase (NRPS) genes were used to amplify gene fragments from a cereulide-producing B. cereus strain. Sequence analysis of one of the amplicons revealed a DNA fragment whose putative gene product showed significant homology to valine activation NRPS modules. The sequences of the flanking regions of this DNA fragment revealed a complete module that is predicted to activate valine, as well as a putative carboxyl-terminal thioesterase domain of the NRPS gene. Disruption of the peptide synthetase gene by insertion of a kanamycin cassette through homologous recombination produced cereulide-deficient mutants. The valine-activating module was highly conserved when sequences from nine emetic B. cereus strains isolated from diverse geographical locations were compared. Primers were designed based on the NRPS sequence, and the resulting PCR assay, targeting the ces gene, was tested by using a panel of 143 B. cereus group strains and 40 strains of other bacterial species showing PCR bands specific for only the cereulide-producing B. cereus strains.     

增加复制起始区对基因表达的影响

我们在质粒ｐｕＢ１１０的基础上组建了ｐＤＲ质粒,它们具有双复制起始区而只有一个抗卡那霉素基因。携带了这些质粒的宿主细胞对卡那霉素的抗性明显高于亲本株（Ｂ,ｓｕｂｔｉｌｉｓ １５０（ｐｕＢ１１０））。经限制性酶切图谱分析新获得的转化株具有二个复制起始区及一个 Ｋｍ＆４基因。说明增加复制起始区是提高重组子表达能力的途径之一。     

Characteristics of Klebsiella pneumoniae plasmid bearing the markers of drug resistance and antilysozyme activity

Electrophoretic study of the profile of plasmid DNA in agarose gel has shown the presence of a plasmid with a molecular weight of 55-60 MD in K. pneumoniae strains possessing antilysozyme activity. Plasmid pAlz60 of K. pneumoniae 22-110, isolated from the blood of a septicemia patient, is a fi- type conjugative plasmid. This plasmid is transferred to recipient strains of different species of enterobacteria with a frequency of 1 X 10(-5) to 1 X 10(-7). Simultaneously with the transfer of the plasmid, recipient cells inherit the antilysozyme markers and resistance to a number of drugs. The discovered plasmid has one restriction site for each of endonucleases EcoRI and XhoI and 16-20 sites for restrictases KpNI, BglII and Hind III.     

Homologous and heterologous transcription of the Cry+-plasmid in Bacillus thuringiensis

The possibility of homologous and heterologous transception of Cry+ plasmids in Bacillus thuringiensis is demonstrated. Cry+ plasmids from crystal bearing strain of Bacillus thuringiensis were transferred into acrystalline strain belonging to H5 serotype by mutual incubation. The donor strain was previously marked by the transmissive plasmid pAM beta 1 coding for erythromycin and lincomycin resistance. The transcipients having acquired the ability to synthesize delta-endotoxin were referred to H5 serotype due to their phenotype. By analogous method Cry+ plasmid was transferred from Bacillus thuringiensis to Bacillus cereus. Bacillus cereus strain GP7 was used as a recipient strain resistant to tetracycline. The presence of delta-endotoxin in transcipients was confirmed by bioprobes and immunoenzyme assay. To prove the transfer of Cry+ plasmid the plasmid profiles of the parent strains and transcipients have been analyzed. The formation of cellular contacts during mutual incubation of Bacillus thuringiensis and Bacillus cereus strains was demonstrated by electron microscopic study of ultrafine cuts.     

Development of a host-vector system in a Rhodococcus strain and its use for expression of the cloned nitrile hydratase gene cluster.

Two different types of plasmid were isolated from strains of Rhodococcus rhodochrous. Two plasmids, of the same type but from different strains, were combined with Escherichia coli plasmids carrying antibiotic resistance markers to develop E. coli-Rhodococcus shuttle vectors. The ampicillin and kanamycin resistance markers served for selection in Rhodococcus. Electroporation was used to introduce recombinant plasmid DNA into R. rhodochrous ATCC 12674 at a frequency of 5 x 10(7) transformants per microgram DNA. With these host-vector and transformation systems, the nitrile hydratase and amidase genes of a Rhodococcus strain were introduced into the host strain and were efficiently expressed.