Characterization of two conjugative Yersinia plasmids mobilizing pYV.
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Chromosome Mapping↗
Biomedical subjects
Bernd Appel
Publications and source records attributed to Bernd Appel.
PY54, a linear plasmid prophage of Yersinia enterocolitica with covalently closed ends.
PY54 is a temperate phage isolated from Yersinia enterocolitica. Lysogenic Yersinia strains harbour the PY54 prophage as a plasmid (pY54). The plasmid has the same size (46 kb) as the PY54 genome isolated from phage particles. By electron microscopy, restriction analysis and DNA sequencing, it was demonstrated that the phage and the plasmid DNAs are linear, circularly permuted molecules. Unusually for phages of Gram-negative bacteria, the phage genome has 3'-protruding ends. The linear plasmid pY54 has covalently closed ends forming telomere-like hairpins. The equivalent DNA sequence of the phage genome is a 42 bp perfect palindrome. Downstream from the palindrome, an open reading frame (ORF) was identified that revealed strong DNA homology to the telN gene of Escherichia coli phage N15 encoding a protelomerase. Similar to PY54, the N15 prophage is a linear plasmid with telomeres. The N15 protelomerase has cleaving/joining activity generating the telomeres by processing a 56 bp palindrome (telomere resolution site tel RL). To study the activity of the PY54 protein, the telN-like gene was cloned and expressed in E. coli. A 77 kDa protein was obtained and partially purified. The protein was found to process recombinant plasmids containing the 42 bp palindrome. Telomere resolution of plasmids under in vivo conditions was also investigated in Yersinia infected with PY54. Processing required a plasmid containing the palindrome as well as adjacent DNA sequences from the phage including an additional inverted repeat. Regions on the phage genome important for plasmid maintenance were defined by the construction of linear and circular miniplasmid derivatives of pY54, of which the smallest miniplasmid comprises a 4.5 kb DNA fragment of the plasmid prophage.
Amino Acid Sequence↗
A cryptic plasmid of Yersinia enterocolitica encodes a conjugative transfer system related to the regions of CloDF13 Mob and IncX Pil.
Yersinia enterocolitica 29930 (biotype 1A; O : 7,8), the producing strain of the phage-tail-like bacteriocin enterocoliticin, possesses a plasmid-encoded conjugative type IV transfer system. The genes of the conjugative system were found by screening of a cosmid library constructed from total DNA of strain 29930. The cosmid Cos100 consists of the vector SuperCos1 and an insert DNA of 40 303 bp derived from a cryptic plasmid of strain 29930. The conjugative transfer system consists of genes encoding a DNA transfer and replication system (Dtr) with close relationship to the mob region of the mobilizable plasmid CloDF13 and a gene cluster encoding a mating pair formation system (Mpf) closely related to the Mpf system of the IncX plasmid R6K. However, a gene encoding a homologue of TaxB, the coupling protein of the IncX system, is missing. The whole transfer region has a size of approximately 17 kb. The recombinant plasmid Cos100 was shown to be transferable between Escherichia coli and Yersinia with transfer frequencies up to 0.1 transconjugants per donor. Mutations generated by inserting a tetracycline cassette into putative tri genes yielded a transfer-deficient phenotype. Conjugative transfer of the cryptic plasmid could not be demonstrated in the original host Y. enterocolitica 29930. However, a kanamycin-resistance-conferring derivative of the plasmid was successfully introduced into E. coli K-12 by transformation and was shown to be self-transmissible. Furthermore, Southern blot hybridization and PCR experiments were carried out to elucidate the distribution of the conjugative transfer system in Yersinia. In total, six Y. enterocolitica biotype 1A strains harbouring closely related systems on endogenous plasmids were identified.
Base Sequence↗
Nucleotide sequence of the integration site of the temperate bacteriophage 6220, which carries the Shiga toxin gene stx(1ox3).
The integration site, attR, of the Shiga toxin-encoding phage 6220 (stx(1ox3)) has been determined. The phage integrates into the chromosome of its Escherichia coli host strain, CB6220, within a gene that is homologous to gene Z2577 and encodes an oxidoreductase. This new integration site was found in different Stx1ox3-producing enterohemorrhagic E. coli strains, which were analyzed by PCR.
Base Sequence↗
Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by real-time PCR.
Bacillus anthracis spores have been shown to be an efficient biological weapon and their recent use in bioterrorist attacks has demonstrated the need for rapid and specific diagnostics. A TaqMan real-time PCR for identification of B. anthracis was developed, based on the two plasmids, pX01 and pX02, both of which are necessary for pathogenicity, as well as on the chromosomally encoded rpoB gene. Bacteria picked from colonies or pelleted from liquid cultures were directly inoculated into the PCR mix, thus avoiding time-consuming DNA preparation and minimizing handling risks. B. anthracis spores were cultivated for a few hours in enrichment broth before PCR analysis, or used directly for real-time PCR, thus allowing to confirm or exclude potential attacks approximately 2-3 h after the material has arrived in the laboratory.
Anthrax↗
Plant-specific promoter sequences carry elements that are recognised by the eubacterial transcription machinery.
During evolution the promoter elements from prokaryotes and eukaryotes have developed differently with regard to their sequence and structure, implying that in general a transfer of eukaryotic promoter sequences into prokaryotes will not cause an efficient gene expression. However, there have been reports on the functionality of the 35S promoter from cauliflower mosaic virus (CaMV) in bacteria. We therefore decided to experimentally investigate the capability of plant promoter sequences to direct gene expression in various bacteria. Accordingly, we tested ten different plant-specific promoters from Solanum tuberosum, Nicotiana tabacum, CaMV, Agrobacterium tumefaciens, and A. rhizogenes for their ability to initiate transcription in five different eubacterial species (Escherichia coli, Yersinia enterocolitica, A. tumefaciens, Pseudomonas putida, and Acinetobacter sp. BD413). To monitor the strength of the plant-specific promoters in bacteria we created fusions between these promoters and the coding region of the luciferase genes from Vibrio harveyi and measured the luminescence in the bacteria. Heterologous gene expression was observed in 50% of the combinations analysed. We then mapped the transcription start site caused by one of the plant-specific promoters, the ST-LS1 promoter from S. tuberosum, in these bacterial species. The location of the mapped transcription start site indicated that the sequences of the plant promoter themselves were recognised by the bacterial transcription apparatus. The recognition of plant-specific promoter sequences by the bacterial RNA polymerase was further confirmed by site-directed mutagenesis of the ST-LS1 promoter and the analysis of the effects of the mutations on the strength of gene expression in E. coli. Using these mutants in our reporter assays we could localise the sequences of the ST-LS1 promoter serving as -10 region in E. coli. The results of our study show that promoter sequences are much less specific than is generally assumed. This is of great importance for our knowledge about the evolution of gene expression systems and for the construction of optimised expression vectors.
Bacteria↗