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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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Occurrence of the deletion derivatives of SGI1 in the populations of the 17 original S. T. DT104 strains harbouring wt SGI1.PCRs were carried out using total DNA of the original strains and of their derivatives harbouring plasmid R55. Parts A, B, C and D: PCR detection of A-, S-, d1- and dflo-type deletion derivatives, respectively. In part C, the first panel shows the wt PCR amplicon. Note that in part D, the wt flo and dflo amplicons appear together in the same PCRs. Part E: Semiquantitative PCR test using 16S rDNA specific primers 16Sfor and 16Srev. PCRs contained templates from ST1233, ST1388 and ST1579 including both the R55- and R55+ series. Cycle numbers applied are shown above the lanes.
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pone-0032497-g005: Occurrence of the deletion derivatives of SGI1 in the populations of the 17 original S. T. DT104 strains harbouring wt SGI1.PCRs were carried out using total DNA of the original strains and of their derivatives harbouring plasmid R55. Parts A, B, C and D: PCR detection of A-, S-, d1- and dflo-type deletion derivatives, respectively. In part C, the first panel shows the wt PCR amplicon. Note that in part D, the wt flo and dflo amplicons appear together in the same PCRs. Part E: Semiquantitative PCR test using 16S rDNA specific primers 16Sfor and 16Srev. PCRs contained templates from ST1233, ST1388 and ST1579 including both the R55- and R55+ series. Cycle numbers applied are shown above the lanes.

Mentions: Our observation that several S- and A-type deletion variant could be isolated even after the first passages of SGI1+S. T. strains suggested that these forms were continuously present as a small fraction in the bacterial populations harbouring wt SGI1. The additional observation that d1 deletion was only detectable in the presence of R55 hinted at the potential role of the helper plasmid in the generation of deletions. To test these possibilities, extensive PCR analysis were carried out for all the 17 original SGI1+S. T. strains and their derivatives harbouring R55. Total DNA was isolated from overnight cultures grown without any selection for SGI1 markers and used in PCRs specific for the four deletions described above (Fig. 5). PCRs were carried out with equal amount of template DNA according to the standard conditions (see Materials and Methods). The results confirmed that the deletion variants were present in most of the populations (except d1, which could not be detected in any of the 17 samples) (Fig. 5A–D), although their proportion was obviously very low. The semiquantitative PCR test for the 16S rDNA demonstrated that different intensities (or absence) of PCR amplicons specific for the deletion derivatives were not related to different DNA concentration of the preparations (Fig. 5E). On the other hand, the presence of R55 in the S. T. strains caused a striking increase in the intensity of PCR signals for all four deletions in the majority of samples. This effect was most significant in the cases of A-, S- and d1-type deletions.


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

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

Occurrence of the deletion derivatives of SGI1 in the populations of the 17 original S. T. DT104 strains harbouring wt SGI1.PCRs were carried out using total DNA of the original strains and of their derivatives harbouring plasmid R55. Parts A, B, C and D: PCR detection of A-, S-, d1- and dflo-type deletion derivatives, respectively. In part C, the first panel shows the wt PCR amplicon. Note that in part D, the wt flo and dflo amplicons appear together in the same PCRs. Part E: Semiquantitative PCR test using 16S rDNA specific primers 16Sfor and 16Srev. PCRs contained templates from ST1233, ST1388 and ST1579 including both the R55- and R55+ series. Cycle numbers applied are shown above the lanes.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0032497-g005: Occurrence of the deletion derivatives of SGI1 in the populations of the 17 original S. T. DT104 strains harbouring wt SGI1.PCRs were carried out using total DNA of the original strains and of their derivatives harbouring plasmid R55. Parts A, B, C and D: PCR detection of A-, S-, d1- and dflo-type deletion derivatives, respectively. In part C, the first panel shows the wt PCR amplicon. Note that in part D, the wt flo and dflo amplicons appear together in the same PCRs. Part E: Semiquantitative PCR test using 16S rDNA specific primers 16Sfor and 16Srev. PCRs contained templates from ST1233, ST1388 and ST1579 including both the R55- and R55+ series. Cycle numbers applied are shown above the lanes.
Mentions: Our observation that several S- and A-type deletion variant could be isolated even after the first passages of SGI1+S. T. strains suggested that these forms were continuously present as a small fraction in the bacterial populations harbouring wt SGI1. The additional observation that d1 deletion was only detectable in the presence of R55 hinted at the potential role of the helper plasmid in the generation of deletions. To test these possibilities, extensive PCR analysis were carried out for all the 17 original SGI1+S. T. strains and their derivatives harbouring R55. Total DNA was isolated from overnight cultures grown without any selection for SGI1 markers and used in PCRs specific for the four deletions described above (Fig. 5). PCRs were carried out with equal amount of template DNA according to the standard conditions (see Materials and Methods). The results confirmed that the deletion variants were present in most of the populations (except d1, which could not be detected in any of the 17 samples) (Fig. 5A–D), although their proportion was obviously very low. The semiquantitative PCR test for the 16S rDNA demonstrated that different intensities (or absence) of PCR amplicons specific for the deletion derivatives were not related to different DNA concentration of the preparations (Fig. 5E). On the other hand, the presence of R55 in the S. T. strains caused a striking increase in the intensity of PCR signals for all four deletions in the majority of samples. This effect was most significant in the cases of A-, S- and d1-type deletions.

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