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Excision of an unstable pathogenicity island in Salmonella enterica serovar Enteritidis is induced during infection of phagocytic cells.

Quiroz TS, Nieto PA, Tobar HE, Salazar-Echegarai FJ, Lizana RJ, Quezada CP, Santiviago CA, Araya DV, Riedel CA, Kalergis AM, Bueno SM - PLoS ONE (2011)

Bottom Line: Importantly, we also found that one type of excision occurred at higher rates when S.Enteritidis was residing inside murine phagocytic cells.These data suggest that ROD21 is an unstable pathogenicity island, whose frequency of excision depends on the environmental conditions found inside phagocytic cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Millennium Institute on Immunology and Immunotherapy, Pontificia Universidad Católica de Chile, Santiago, Chile.

ABSTRACT
The availability of the complete genome sequence of several Salmonella enterica serovars has revealed the presence of unstable genetic elements in these bacteria, such as pathogenicity islands and prophages. This is the case of Salmonella enterica serovar Enteritidis (S. Enteritidis), a bacterium that causes gastroenteritis in humans and systemic infection in mice. The whole genome sequence analysis for S. Enteritidis unveiled the presence of several genetic regions that are absent in other Salmonella serovars. These regions have been denominated "regions of difference" (ROD). In this study we show that ROD21, one of such regions, behaves as an unstable pathogenicity island. We observed that ROD21 undergoes spontaneous excision by two independent recombination events, either under laboratory growth conditions or during infection of murine cells. Importantly, we also found that one type of excision occurred at higher rates when S. Enteritidis was residing inside murine phagocytic cells. These data suggest that ROD21 is an unstable pathogenicity island, whose frequency of excision depends on the environmental conditions found inside phagocytic cells.

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ROD21 excision can be generated by means of two different recombination events.Amplification of attB and attP sequences generated after type 1 and type 2 excisions were detected by nested PCR in LK5, PT4, PT1 and PT21 strains of S. Enteritidis, using primer pairs described in Table 2 and in Figure 2. PCR products for attB (A) and attP (B) sequences for each type of excision were resolved in 1% agarose gels. The sequence of each PCR product was obtained (chromatograms in each Figure) and compared with the attB and attP sequences deduced for type 1 and type 2 excisions (labeled as theoretical). attB and attP sequences are highlighted in red in both alignments and chromatograms. Expected size for each PCR product: 591 bp for type 1 excision attB, 657 bp for type 2 excision attB, 995 bp for type 1 excision attP and 1050 bp for type 2 excision attP.
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pone-0026031-g003: ROD21 excision can be generated by means of two different recombination events.Amplification of attB and attP sequences generated after type 1 and type 2 excisions were detected by nested PCR in LK5, PT4, PT1 and PT21 strains of S. Enteritidis, using primer pairs described in Table 2 and in Figure 2. PCR products for attB (A) and attP (B) sequences for each type of excision were resolved in 1% agarose gels. The sequence of each PCR product was obtained (chromatograms in each Figure) and compared with the attB and attP sequences deduced for type 1 and type 2 excisions (labeled as theoretical). attB and attP sequences are highlighted in red in both alignments and chromatograms. Expected size for each PCR product: 591 bp for type 1 excision attB, 657 bp for type 2 excision attB, 995 bp for type 1 excision attP and 1050 bp for type 2 excision attP.

Mentions: To evaluate whether these potential recombinations can occur, we performed a PCR reaction using primers that hybridize upstream and downstream of asnT genes and the DRS. As shown in Fig. 2B and 2C, these hypothetical excision events yield two different attB and attP sequences, which could be detected by PCR using several different primer combinations. To detect these excisions, the genomic DNA of four S. Enteritidis strains was obtained as described in materials and methods and tested by PCR. Conventional PCR amplifications failed to produce measurable amounts of the expected PCR products for each of the attB and attP sequences in each of the S. Enteritidis strains evaluated (data not shown). To increase the sensitivity of detection, nested PCRs were performed as described in material and methods and the expected sized amplicons were obtained: 591 and 657 bp for the chromosomal attB-1 and attB-2 respectively (Fig. 3A) and 958 and 1058 for the episomal attP-1 and attP-2 respectively (Fig. 3B). To corroborate the specificity of these PCR products, the DNA fragments obtained from S. Enteritidis PT1 were sequenced. As shown in Fig. 3, each of the obtained PCR products matched the expected attB (Fig. 3A) and attP (Fig. 3B) sequences. These data suggest that both recombination events occurred at low frequency when bacteria grew to stationary phase in LB medium.


Excision of an unstable pathogenicity island in Salmonella enterica serovar Enteritidis is induced during infection of phagocytic cells.

Quiroz TS, Nieto PA, Tobar HE, Salazar-Echegarai FJ, Lizana RJ, Quezada CP, Santiviago CA, Araya DV, Riedel CA, Kalergis AM, Bueno SM - PLoS ONE (2011)

ROD21 excision can be generated by means of two different recombination events.Amplification of attB and attP sequences generated after type 1 and type 2 excisions were detected by nested PCR in LK5, PT4, PT1 and PT21 strains of S. Enteritidis, using primer pairs described in Table 2 and in Figure 2. PCR products for attB (A) and attP (B) sequences for each type of excision were resolved in 1% agarose gels. The sequence of each PCR product was obtained (chromatograms in each Figure) and compared with the attB and attP sequences deduced for type 1 and type 2 excisions (labeled as theoretical). attB and attP sequences are highlighted in red in both alignments and chromatograms. Expected size for each PCR product: 591 bp for type 1 excision attB, 657 bp for type 2 excision attB, 995 bp for type 1 excision attP and 1050 bp for type 2 excision attP.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198454&req=5

pone-0026031-g003: ROD21 excision can be generated by means of two different recombination events.Amplification of attB and attP sequences generated after type 1 and type 2 excisions were detected by nested PCR in LK5, PT4, PT1 and PT21 strains of S. Enteritidis, using primer pairs described in Table 2 and in Figure 2. PCR products for attB (A) and attP (B) sequences for each type of excision were resolved in 1% agarose gels. The sequence of each PCR product was obtained (chromatograms in each Figure) and compared with the attB and attP sequences deduced for type 1 and type 2 excisions (labeled as theoretical). attB and attP sequences are highlighted in red in both alignments and chromatograms. Expected size for each PCR product: 591 bp for type 1 excision attB, 657 bp for type 2 excision attB, 995 bp for type 1 excision attP and 1050 bp for type 2 excision attP.
Mentions: To evaluate whether these potential recombinations can occur, we performed a PCR reaction using primers that hybridize upstream and downstream of asnT genes and the DRS. As shown in Fig. 2B and 2C, these hypothetical excision events yield two different attB and attP sequences, which could be detected by PCR using several different primer combinations. To detect these excisions, the genomic DNA of four S. Enteritidis strains was obtained as described in materials and methods and tested by PCR. Conventional PCR amplifications failed to produce measurable amounts of the expected PCR products for each of the attB and attP sequences in each of the S. Enteritidis strains evaluated (data not shown). To increase the sensitivity of detection, nested PCRs were performed as described in material and methods and the expected sized amplicons were obtained: 591 and 657 bp for the chromosomal attB-1 and attB-2 respectively (Fig. 3A) and 958 and 1058 for the episomal attP-1 and attP-2 respectively (Fig. 3B). To corroborate the specificity of these PCR products, the DNA fragments obtained from S. Enteritidis PT1 were sequenced. As shown in Fig. 3, each of the obtained PCR products matched the expected attB (Fig. 3A) and attP (Fig. 3B) sequences. These data suggest that both recombination events occurred at low frequency when bacteria grew to stationary phase in LB medium.

Bottom Line: Importantly, we also found that one type of excision occurred at higher rates when S.Enteritidis was residing inside murine phagocytic cells.These data suggest that ROD21 is an unstable pathogenicity island, whose frequency of excision depends on the environmental conditions found inside phagocytic cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Millennium Institute on Immunology and Immunotherapy, Pontificia Universidad Católica de Chile, Santiago, Chile.

ABSTRACT
The availability of the complete genome sequence of several Salmonella enterica serovars has revealed the presence of unstable genetic elements in these bacteria, such as pathogenicity islands and prophages. This is the case of Salmonella enterica serovar Enteritidis (S. Enteritidis), a bacterium that causes gastroenteritis in humans and systemic infection in mice. The whole genome sequence analysis for S. Enteritidis unveiled the presence of several genetic regions that are absent in other Salmonella serovars. These regions have been denominated "regions of difference" (ROD). In this study we show that ROD21, one of such regions, behaves as an unstable pathogenicity island. We observed that ROD21 undergoes spontaneous excision by two independent recombination events, either under laboratory growth conditions or during infection of murine cells. Importantly, we also found that one type of excision occurred at higher rates when S. Enteritidis was residing inside murine phagocytic cells. These data suggest that ROD21 is an unstable pathogenicity island, whose frequency of excision depends on the environmental conditions found inside phagocytic cells.

Show MeSH
Related in: MedlinePlus