Broad-host-range Yersinia phage PY100: genome sequence, proteome analysis of virions, and DNA packaging strategy.

PY100 is a lytic bacteriophage with a broad host range within the genus Yersinia. The phage forms plaques on strains of the three human pathogenic species Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis at 37 degrees C. PY100 was isolated from farm manure and intended to be used in phage therapy trials. PY100 has an icosahedral capsid containing double-stranded DNA and a contractile tail. The genome consists of 50,291 bp and is predicted to contain 93 open reading frames (ORFs). PY100 gene products were found to be homologous to the capsid proteins and proteins involved in DNA metabolism of the enterobacterial phage T1; PY100 tail proteins possess homologies to putative tail proteins of phage AaPhi23 of Actinobacillus actinomycetemcomitans. In a proteome analysis of virion particles, 15 proteins of the head and tail structures were identified by mass spectrometry. The putative gene product of ORF2 of PY100 shows significant homology to the gene 3 product (small terminase subunit) of Salmonella phage P22 that is involved in packaging of the concatemeric phage DNA. The packaging mechanism of PY100 was analyzed by hybridization and sequence analysis of DNA isolated from virion particles. Newly replicated PY100 DNA is cut initially at a pac recognition site, which is located in the coding region of ORF2.

Isolation of Vibrio vulnificus and atypical Vibrio from surface water of the Baltic Sea in Germany.

From 1995 to 1997 several defined species like V. alginolyticus, V. anguillarum, V. cholerae (non O1 and non O139), V. mimicus, V. parahaemolyticus and V. vulnificus were isolated during a survey to determine the presence of V. vulnificus in the brackish water of the Baltic Sea in Germany. Moreover atypical Vibrio isolates were detected. Four isolates belonging to a group of atypical Vibrio and possibly representing a new species in the genus Vibrio were characterized in detail. All four strains were isolated from surface costal waters. Based on 16S rDNA sequence analysis they showed the highest relatedness to the species V. navarrensis and V. vulnificus. The strains did not harbor the species specific hemolysin gene vvhA from V. vulnificus as shown by PCR and hybridization experiments. Moreover, they differed in at least two biochemical parameters tested from the hitherto described Vibrio species. All these strains induced hemolysis on washed blood agar dishes and showed phase variations on Luria Bertani agar dishes. Because of the similarity to the eel pathogen V. vulnificus, we infected eels with one of the four atypical strains (CH-291), but no pathogenicity for eels could be detected. Furthermore, Vero cell tests with supernatants of bacterial cultures did not reveal secreted Vero cell cytotoxic compounds. This indicates a nonpathogenic nature of these strains.

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.