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Stability, entrapment and variant formation of Salmonella genomic island 1.

Kiss J, Nagy B, Olasz F - PLoS ONE (2012)

Bottom Line: Here we provide experimental studies on the stability, entrapment and variant formation of this peculiar gene cluster originally found in S.Typhimurium.SGI1-free segregants did not accumulate during long-term propagation, but several SGI1 variants could be obtained.

View Article: PubMed Central - PubMed

Affiliation: Agricultural Biotechnology Center, Gödöllő, Hungary. kissj@abc.hu

ABSTRACT

Background: The Salmonella genomic island 1 (SGI1) is a 42.4 kb integrative mobilizable element containing several antibiotic resistance determinants embedded in a complex integron segment In104. The numerous SGI1 variants identified so far, differ mainly in this segment and the explanations of their emergence were mostly based on comparative structure analyses. Here we provide experimental studies on the stability, entrapment and variant formation of this peculiar gene cluster originally found in S. Typhimurium.

Methodology/principal findings: Segregation and conjugation tests and various molecular techniques were used to detect the emerging SGI1 variants in Salmonella populations of 17 Salmonella enterica serovar Typhimurium DT104 isolates from Hungary. The SGI1s in these isolates proved to be fully competent in excision, conjugal transfer by the IncA/C helper plasmid R55, and integration into the E. coli chromosome. A trap vector has been constructed and successfully applied to capture the island on a plasmid. Monitoring of segregation of SGI1 indicated high stability of the island. SGI1-free segregants did not accumulate during long-term propagation, but several SGI1 variants could be obtained. Most of them appeared to be identical to SGI1-B and SGI1-C, but two new variants caused by deletions via a short-homology-dependent recombination process have also been detected. We have also noticed that the presence of the conjugation helper plasmid increased the formation of these deletion variants considerably.

Conclusions/significance: Despite that excision of SGI1 from the chromosome was proven in SGI1(+)Salmonella populations, its complete loss could not be observed. On the other hand, we demonstrated that several variants, among them two newly identified ones, arose with detectable frequencies in these populations in a short timescale and their formation was promoted by the helper plasmid. This reflects that IncA/C helper plasmids are not only involved in the horizontal spreading of SGI1, but may also contribute to its evolution.

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Sequence chart of dflo PCR amplicon.The 304 bp dflo PCR fragment obtained from total DNA of strain ST1375 using primers flofor and florev was isolated from agarose gel and directly sequenced with primer flofor. The alignment of floDRL and floDRR are shown below the chart, coordinates are indicated according to the SGI1 sequence (GenBank AF261825.2). The nine mismatched positions and the corresponding signal in the primary sequence are indicated by arrows.
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pone-0032497-g004: Sequence chart of dflo PCR amplicon.The 304 bp dflo PCR fragment obtained from total DNA of strain ST1375 using primers flofor and florev was isolated from agarose gel and directly sequenced with primer flofor. The alignment of floDRL and floDRR are shown below the chart, coordinates are indicated according to the SGI1 sequence (GenBank AF261825.2). The nine mismatched positions and the corresponding signal in the primary sequence are indicated by arrows.

Mentions: The formation of d1 deletion by recombination between short direct repeats raised the possibility that a similar process using other direct repeats in SGI1 can lead to additional deletion variants. Since the florfenicol resistance gene floR is delimited by 102-bp imperfect repeats (floDRL and floDRR, Fig. 2A), this region appeared to be suitable to test this assumption. PCR primers (flofor-florev) specific for the outer flanking regions of these repeats were designed and the SGI1+ ST1375 strain and its derivative harbouring the IncA/C helper plasmid IP40a were tested for the appearance of the variant deleted for floR (dflo). PCRs resulted in two fragments and sequencing proved that the large fragment (1945 bp) corresponded to the wt situation, while the 304 bp fragment was amplified from dflo templates (Fig. 2D). The appearance of the alternative bases in the sequence of the 304 bp PCR amplicon at the 9 positions, where floDRL and floDRR are mismatched (Fig. 4), suggested that crossing over could have occurred at many positions along the 102-bp homology, thus the resulting subpopulation of dflo variants may represent independent recombination events. Since d1 derivatives were detected in the presence of R55 conferring resistance for Chm/Flo, Kan, Amp, Gen and Sul, instead, a close relative helper plasmid, IP40a, conferring resistance only for Kan, Amp and Sul was applied to isolate dflo (AmpRStrRSptRTetRSulRChmS) SGI1 derivatives. IP40a was previously shown to participate in SGI1 mobilization similarly to R55 [20]. Two parallel colonies of strains ST1375 and ST1375/IP40a were grown in LB until the stationary phase under selection for Tet, but without selection for Chm (this prevented the accumulation of A- and S-type derivatives, which are also ChmS). The dflo derivatives then were sought by replica plating. Although PCR and sequencing results clearly showed the presence of dflo derivatives in the bacterial populations, no ChmSTetR segregants could be isolated among 9723 and 10420 TetR colonies of the two strains. This suggested that the frequency of dflo variants in the stationary phase populations was <10−4 per TetR cells.


Stability, entrapment and variant formation of Salmonella genomic island 1.

Kiss J, Nagy B, Olasz F - PLoS ONE (2012)

Sequence chart of dflo PCR amplicon.The 304 bp dflo PCR fragment obtained from total DNA of strain ST1375 using primers flofor and florev was isolated from agarose gel and directly sequenced with primer flofor. The alignment of floDRL and floDRR are shown below the chart, coordinates are indicated according to the SGI1 sequence (GenBank AF261825.2). The nine mismatched positions and the corresponding signal in the primary sequence are indicated by arrows.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285670&req=5

pone-0032497-g004: Sequence chart of dflo PCR amplicon.The 304 bp dflo PCR fragment obtained from total DNA of strain ST1375 using primers flofor and florev was isolated from agarose gel and directly sequenced with primer flofor. The alignment of floDRL and floDRR are shown below the chart, coordinates are indicated according to the SGI1 sequence (GenBank AF261825.2). The nine mismatched positions and the corresponding signal in the primary sequence are indicated by arrows.
Mentions: The formation of d1 deletion by recombination between short direct repeats raised the possibility that a similar process using other direct repeats in SGI1 can lead to additional deletion variants. Since the florfenicol resistance gene floR is delimited by 102-bp imperfect repeats (floDRL and floDRR, Fig. 2A), this region appeared to be suitable to test this assumption. PCR primers (flofor-florev) specific for the outer flanking regions of these repeats were designed and the SGI1+ ST1375 strain and its derivative harbouring the IncA/C helper plasmid IP40a were tested for the appearance of the variant deleted for floR (dflo). PCRs resulted in two fragments and sequencing proved that the large fragment (1945 bp) corresponded to the wt situation, while the 304 bp fragment was amplified from dflo templates (Fig. 2D). The appearance of the alternative bases in the sequence of the 304 bp PCR amplicon at the 9 positions, where floDRL and floDRR are mismatched (Fig. 4), suggested that crossing over could have occurred at many positions along the 102-bp homology, thus the resulting subpopulation of dflo variants may represent independent recombination events. Since d1 derivatives were detected in the presence of R55 conferring resistance for Chm/Flo, Kan, Amp, Gen and Sul, instead, a close relative helper plasmid, IP40a, conferring resistance only for Kan, Amp and Sul was applied to isolate dflo (AmpRStrRSptRTetRSulRChmS) SGI1 derivatives. IP40a was previously shown to participate in SGI1 mobilization similarly to R55 [20]. Two parallel colonies of strains ST1375 and ST1375/IP40a were grown in LB until the stationary phase under selection for Tet, but without selection for Chm (this prevented the accumulation of A- and S-type derivatives, which are also ChmS). The dflo derivatives then were sought by replica plating. Although PCR and sequencing results clearly showed the presence of dflo derivatives in the bacterial populations, no ChmSTetR segregants could be isolated among 9723 and 10420 TetR colonies of the two strains. This suggested that the frequency of dflo variants in the stationary phase populations was <10−4 per TetR cells.

Bottom Line: Here we provide experimental studies on the stability, entrapment and variant formation of this peculiar gene cluster originally found in S.Typhimurium.SGI1-free segregants did not accumulate during long-term propagation, but several SGI1 variants could be obtained.

View Article: PubMed Central - PubMed

Affiliation: Agricultural Biotechnology Center, Gödöllő, Hungary. kissj@abc.hu

ABSTRACT

Background: The Salmonella genomic island 1 (SGI1) is a 42.4 kb integrative mobilizable element containing several antibiotic resistance determinants embedded in a complex integron segment In104. The numerous SGI1 variants identified so far, differ mainly in this segment and the explanations of their emergence were mostly based on comparative structure analyses. Here we provide experimental studies on the stability, entrapment and variant formation of this peculiar gene cluster originally found in S. Typhimurium.

Methodology/principal findings: Segregation and conjugation tests and various molecular techniques were used to detect the emerging SGI1 variants in Salmonella populations of 17 Salmonella enterica serovar Typhimurium DT104 isolates from Hungary. The SGI1s in these isolates proved to be fully competent in excision, conjugal transfer by the IncA/C helper plasmid R55, and integration into the E. coli chromosome. A trap vector has been constructed and successfully applied to capture the island on a plasmid. Monitoring of segregation of SGI1 indicated high stability of the island. SGI1-free segregants did not accumulate during long-term propagation, but several SGI1 variants could be obtained. Most of them appeared to be identical to SGI1-B and SGI1-C, but two new variants caused by deletions via a short-homology-dependent recombination process have also been detected. We have also noticed that the presence of the conjugation helper plasmid increased the formation of these deletion variants considerably.

Conclusions/significance: Despite that excision of SGI1 from the chromosome was proven in SGI1(+)Salmonella populations, its complete loss could not be observed. On the other hand, we demonstrated that several variants, among them two newly identified ones, arose with detectable frequencies in these populations in a short timescale and their formation was promoted by the helper plasmid. This reflects that IncA/C helper plasmids are not only involved in the horizontal spreading of SGI1, but may also contribute to its evolution.

Show MeSH
Related in: MedlinePlus