1 Hokkaido Research Station, National Institute of Animal Health, Hitsujigaoka-4, Toyohira, Sapporo 062-0045, Japan
2 Nemuro Livestock Hygiene Service Center, Betsukaimidorimachi-69, Betsukai, Notsukegun 086-0214, Japan
3 Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro 080-8555, Japan
4 National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan
Correspondence
Ikuo Uchida
ikuouchi{at}affrc.go.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AB104436.
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INTRODUCTION |
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In the present study, we have identified another prophage-like element in the supernatant of a S. typhimurium DT104 isolate treated with mitomycin C. Genome walking analysis revealed the presence of putative pertussis-like toxin genes in the flanking region of the prophage-like element. Here we show the distribution of this novel gene among S. typhimurium DT104 isolates.
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METHODS |
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Induction of lysogen.
In order to identify putative lysogenic phages, the S. typhimurium DT104 strain U1 was grown overnight in 10 ml LB broth, before subculturing at 1 : 10 into 50 ml culture with shaking. After the early exponential phase, mitomycin C was added at a concentration of 0·5 µg ml1 and the mixture incubated for a further 16 h with vigorous shaking as previously described (Tanaka et al., 2004; Yee et al., 1993
). The bacterial cells were removed by centrifugation (9800 g, 10 min) and filtered through a 0·45 µm membrane filter. After digesting bacterial DNAs and RNAs by DNase (100 µg ml1) and RNase (50 µg ml1) for 2 h at 37 °C, phage particles were precipitated with 10 % (w/v) PEG 6000 and 1 M NaCl following the methods described by Ikebe et al. (2002)
. DNAs in the particles were then extracted by phenol and precipitated with ethanol.
Cloning and DNA sequencing.
DNA sequencing was performed with an automated DNA sequencer (ABI Prism 377; Perkin-Elmer Applied Biosystems) by using an ABI Prism BigDye Terminator Cycle Sequencing Kit (Perkin-Elmer Applied Biosystems). The 0·4 kb HindIII fragment of a putative phage genome was cloned in pBluescript II and the initial sequence information of the clone was obtained with universal and reverse primers for pUC/M13 vectors. This sequence was used to create customized oligonucleotides for primer walking.
Then adjacent DNA sequences upstream and downstream of the 0·4 kb HindIII fragment in the U1 genome were investigated with a Clontech Universal Genome Walker Kit. Details of the methodology used can be found in the user manual at http://www.clontech.com/. Briefly, U1 genomic DNA was digested with one of four different blunt-ended restriction enzymes provided by the manufacturer. The purified restricted DNA fragments were ligated to an adaptor supplied by the manufacturer and became a part of four genome-walking libraries generated by the four different restriction enzymes. Specific primers based on the sequence of the 0·4 kb HindIII fragment were designed and used in combination with adaptor-specific primers to amplify the large genomic segments adjacent to the 0·4 kb HindIII fragment by long-distance PCR using a Takara LA PCR Kit in a 9700 thermal cycler (Applied Biosystems) as described by the manufacturer. PCR products were purified with a Qiagen QIAquick PCR Purification Kit for sequencing.
PCR.
Standard PCR was carried out in a mixture of 50 µl containing 1xPCR buffer with 0·2 mM MgCl2, 2·5 U AmpliTaq DNA polymerase, 0·2 mM deoxynucleotide triphosphates, 0·5 µM primers and 50 ng genomic DNA. PCR amplification was performed with the first denaturation cycle at 95 °C for 1 min, followed by 30 cycles, each consisting of 30 s denaturation at 94 °C and 30 s annealing at 55 °C, and 1 min extension at 72 °C. Incubation for 3 min at 72 °C followed to complete extension.
Southern hybridization analysis.
A DNA fragment was amplified with the specific primer set ART-1 (5'-CTGGTTATGCAAGTGCTGTT-3') and ART-2 (5'-CTCCCCGTGCGTCATAAAAC-3'), which amplified a 566 bp internal fragment of artA gene. The PCR product was purified with a QIAquick Gel Extraction Kit (Qiagen) and was labelled with digoxigenin (DIG)-11-dUTP by random priming using a DIG High Prime Labelling Kit (Roche Diagnostics) as described by the manufacturer. Genomic DNA was prepared from S. typhimurium strains by the method previously described (Tamada et al., 2001) and digested with HindIII, separated on 0·8 % (w/v) agarose gel and transferred to a positive membrane (Roche Diagnostics) by the capillary method. Prehybridization (>30 min) and hybridization (>16 h) using Easy Hyb solution (Roche Diagnostics) under high-stringency conditions and digoxigenin detection of hybrids were carried out using a DIG Luminescent Detection Kit (Roche Diagnostics), following the manufacturer's instructions. Hyper MP film (Amersham International) was exposed to membranes for 110 min at room temperature and developed in a Kodak X-Omat processor. PFGE of DNA extracted from the phage particle fraction was performed using 1·2 % (w/v) agarose gel and a 2 s switching interval was applied for 10 h. Subsequent transfer of DNAs and Southern hybridization were carried out as described above.
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RESULTS |
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Presence of artA sequence among S. typhimurium strains
In order to examine the presence of the artA sequence among S. typhimurium isolates, Southern hybridization was performed. A PCR fragment specific to the coding sequence of artA was labelled and used as a probe. HindIII-digested chromosomal DNA from all 12 strains of DT104, and four strains which are not phage-typed but were classified into the same group as DT104 by PFGE and amplified-fragment length polymorphism (AFLP) analysis (Tamada et al., 2001), revealed hybridizing signals at 2·2 kb (Fig. 5
). The same signal was also detected in NCTC 73, a strain isolated from humans in France in 1917, which is classified into a different group from that of DT104 (Tamada et al., 2001
). No intense signals were detected following hybridization of the artA probe with DNA from the 13 other S. typhimurium strains which belonged to groups other than DT104 (Tamada et al., 2001
).
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DISCUSSION |
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artA and artB were identified in adjacent sequences of a 0·4 kb HindIII fragment. The sequence of this 0·4 kb fragment shows homology to a segment of temperate phage ST64B which was isolated from S. typhimurium DT64 (Mmolawa et al., 2003). This phage is induced by mitomycin C, but is not able to produce plaques on a wide range of isolates (Mmolawa et al., 2002
). Furthermore, the flanking region of artAB was found to contain another prophage or putative prophage-like sequences. We examined the inducibility of temperate phage using mitomycin C; however, we were not able to detect plaques of phage without ST104 with indicator strain LT2 by the analysis of plaque hybridization (data not shown). Presence of a stronger signal when the artA probe was hybridized with DNA obtained from a mitomycin C-induced S. typhimurium DT104 strain compared with a non-induced strain suggested that artA resides within a prophage other than ST104. Bacteriophages have played a critical role in the evolution of many bacterial pathogens (Wagner & Waldor, 2002
). Toxins of a number of both Gram-positive and Gram-negative pathogens are encoded in the genomes of temperate bacteriophages. The structural genes encoding diphtheria toxin, cholera toxin and Shiga toxin 1 and 2 are all phage-encoded. The diverse bacteriophages that encode such virulence factors provide a means for the horizontal transmission of virulence genes within a bacterial population. We screened S. typhimurium strains for the presence of artA. Southern blotting analysis of the genomic DNA of S. typhimurium strains with a DIG-labelled probe for artA revealed that artA sequences are present in the genomes of all of the 12 DT104 isolates and four strains which are grouped into the same cluster as DT104 by PFGE and AFLP analysis, irrespective of their origin. The artA sequence was detected in the non-DT104 strain NCTC 73, which was isolated from humans in France in 1917; however, the other 13 S. typhimurium strains were all assumed to lack the artA locus. The results presented here suggest that phage-mediated recombination has played a critical role in acquisition of art genes in S. typhimurium DT104. Since no hybridization signal with an artA probe was observed in the phage particle fraction of strain NCTC 73 (data not shown), artA may not be encoded by the prophage in this strain. The artAB genes in DT104 may have originated from S. typhi, S. paratyphi A or from a strain such as NCTC 73.
Surveillance programme data show that the incidence of salmonellosis in cattle in Japan, especially in adult cattle, has increased continuously since 1992. An increased frequency of isolation of DT104 from cattle has also been noted in Japan since 1992 (Sameshima et al., 2000; Tamada et al., 2001
). Some questions remain about the nature of the possible increased virulence of drug-resistant DT104 strains, since infection by DT104 is more likely to lead to hospitalization for a human or death of cattle than infection by a non-DT104 S. typhimurium (Glynn et al., 1998
). Thus, these case control studies suggest that DT104 may be hypervirulent compared to antibiotic-susceptible strains of S. typhimurium (Glynn et al., 1998
). A study of virulence in S. typhimurium can be performed by assessing the cellular invasion capabilities of individual isolates using a tissue culture assay. However, the ability of DT104 isolates to survive within murine peritoneal macrophages and to invade cultured epithelial cells, and the level of their lethality in mice, have been assessed and failed to demonstrate that DT104 isolates are more virulent than non-DT104 isolates (Allen et al., 2001
). Recently, it was reported that certain strains of DT104 secrete a putative cytotoxin, Clg, which is similar to a collagenase-like protein (Wu et al., 2002
). Clg exerts cytopathic effects that mimic the DT104-mediated cytotoxicosis (Carlson et al., 2001
; Wu et al., 2002
). The clg gene is present in Salmonella and other Gram-negative pathogens even though its transcripts were not detected in vitro (Wu et al., 2002
). Furthermore, the addition of ADP-ribosyltransferase toxin could lead to an apparent enhanced virulence phenotype. In order to answer the questions surrounding the pathogenicity of DT104, further studies are now in progress to determine whether the ADP-ribosyltransferase toxin gene homologue, which is common among DT104 isolates, is expressed, and is active and exported.
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ACKNOWLEDGEMENTS |
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Received 2 February 2005;
revised 12 May 2005;
accepted 20 June 2005.
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