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Secondary chromosomal attachment site and tandem integration of the mobilizable Salmonella genomic island 1.

Doublet B, Golding GR, Mulvey MR, Cloeckaert A - PLoS ONE (2008)

Bottom Line: In the absence of thdF, the frequency of transconjugant formation was reduced by around thirty times of magnitude.At this 2(nd)attB site, we found that a significant fraction of SGI1 transconjugants (43% of wild type and 100% of Delta thdF mutant) contained tandem SGI1 arrays.The existence of a secondary attachment site in the Salmonella chromosome has potential implications for the mobility of SGI1, which may integrate in other attachment sites of other bacterial pathogens that do not possess the 1(st) or 2(nd) specific SGI1 attB sites of Salmonella.

View Article: PubMed Central - PubMed

Affiliation: INRA, UR1282, Infectiologie Animale et Santé Publique, Nouzilly, France. Benoit.Doublet@tours.inra.fr

ABSTRACT

Background: The Salmonella genomic island 1 is an integrative mobilizable element (IME) originally identified in epidemic multidrug-resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) DT104. SGI1 contains a complex integron, which confers various multidrug resistance phenotypes due to its genetic plasticity. Previous studies have shown that SGI1 integrates site-specifically into the S. enterica, Escherichia coli, or Proteus mirabilis chromosome at the 3' end of thdF gene (attB site).

Methodology/principal findings: Here, we report the transfer of SGI1 to a Delta thdF mutant of S. Typhimurium LT2. In the absence of thdF, the frequency of transconjugant formation was reduced by around thirty times of magnitude. Through DNA sequencing SGI1 was shown to integrate specifically into a secondary attachment site (2(nd)attB), which is located in the intergenic region between the chromosomal sodB and purR genes. At this 2(nd)attB site, we found that a significant fraction of SGI1 transconjugants (43% of wild type and 100% of Delta thdF mutant) contained tandem SGI1 arrays. Moreover, in wild type S. Typhimurium LT2 transconjugants, SGI1 integrated into both attachment sites, i.e., thdF and sodB-purR. The formation of SGI1 tandem arrays occurred in both specific attB sites. There was heterogeneity in the size of the SGI1 tandem arrays detected in single transconjugant colonies. Some arrays consisted as far as six SGI1s arranged in tandem. These tandem arrays were shown to persist during serial passages with or without antibiotic selection pressure.

Conclusions/significance: The ability of integration into two distinct chromosomal sites and tandem array formation of SGI1 could contribute to its spread and persistence. The existence of a secondary attachment site in the Salmonella chromosome has potential implications for the mobility of SGI1, which may integrate in other attachment sites of other bacterial pathogens that do not possess the 1(st) or 2(nd) specific SGI1 attB sites of Salmonella.

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Comparison of the SGI1 18 bp attP, attB, DR-Ls, DR-Rs of S. Typhimurium.(A) Alignment of the attP site of SGI1 and the primary attB site (1st attB) of S. Typhimurium strain LT2. The sequence of direct repeats left (DR-L) and right (DR-R) flanking integrated SGI1 were indicated. (B) Alignment of the attP site of SGI1 and the secondary attB site (2nd attB) of S. Typhimurium strain LT2. The sequence of direct repeats left (DR-L) and right (DR-R) were determined in ten independent ΔthdF mutant S. Typhimurium LT2 SGI1 transconjugants from three mating experiments. (*) indicated identical positions in the attP site and the attB sites. Positions 1, 6, 12, and 18 are indicated below the 1st and 2nd attB sequences. The specific nucleotides for attP are underlined in DR-Ls and DR-Rs. Sites of possible cleavage during the strand exchange are indicated by arrows in attP. Nucleotides in boldface letters represent illegitimate base pair in the DR-L and DR-R sequences. The sequences of DR-L and DR-R of some transconjugants revealed a mix of G and A at position 5 in repeated attempts.
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pone-0002060-g002: Comparison of the SGI1 18 bp attP, attB, DR-Ls, DR-Rs of S. Typhimurium.(A) Alignment of the attP site of SGI1 and the primary attB site (1st attB) of S. Typhimurium strain LT2. The sequence of direct repeats left (DR-L) and right (DR-R) flanking integrated SGI1 were indicated. (B) Alignment of the attP site of SGI1 and the secondary attB site (2nd attB) of S. Typhimurium strain LT2. The sequence of direct repeats left (DR-L) and right (DR-R) were determined in ten independent ΔthdF mutant S. Typhimurium LT2 SGI1 transconjugants from three mating experiments. (*) indicated identical positions in the attP site and the attB sites. Positions 1, 6, 12, and 18 are indicated below the 1st and 2nd attB sequences. The specific nucleotides for attP are underlined in DR-Ls and DR-Rs. Sites of possible cleavage during the strand exchange are indicated by arrows in attP. Nucleotides in boldface letters represent illegitimate base pair in the DR-L and DR-R sequences. The sequences of DR-L and DR-R of some transconjugants revealed a mix of G and A at position 5 in repeated attempts.

Mentions: In previous studies, the left and right junctions of SGI1 integrated in the last 18 bp of the thdF gene (named 1st attB site in this study) have been sequenced and analyzed [4], [13], [16]. The sequence of the specific recombinational site (SGI1 attP site) of the extrachromosomal circular form of SGI1 has been also previously determined [13]. Integration of SGI1 in its 1st attB site was shown to occur by recombination mediated by the SGI1 integrase between the 18 bp attP site of the circular form and the 18 bp 1st attB site at the 3′ end of the thdF gene [13]. Compared to the SGI1 attP sequence, the 1st attB site of S. Typhimurium strain LT2 presents two nucleotide substitutions at positions 9 and 12 (Fig. 2A). Analysis of DR-L and DR-R in S. Typhimurium DT104 in which SGI1 is integrated in its 1st attB site demonstrated that these two nucleotide substitutions were always found in the DR-R (Fig. 2A). This result suggests that the cleavage and strand exchange occur somewhere upstream the position 9 during SGI1 integration in its 1st attB site (Fig. 2A). The integration of SGI1 in the 2nd attB site was slightly different. For the 10 ΔthdF mutant S. Typhimurium LT2 transconjugants, the sequences of the left and right junctions were determined to analyze the direct repeat sequences flanking SGI1 in this 2nd attB site (Fig. 2B). As shown in Figure 2B, the sequence of the 2nd attB site in the S. Typhimurium LT2 chromosome differs both in length and sequence from the specific SGI1 attP sequence. Compared to the SGI1 attP site, the 2nd attB site is only 14 bp in length and presents three additional substitutions at positions 3, 5, and 15 to the four gap positions (Fig. 2B). The differences in the SGI1 attP site and the 2nd attB site result in different DR-L and DR-R sequences that allow the cleavage sites during recombination between attP and attB to be estimated (Fig. 2B). The sequences of DR-L and DR-R suggest that one cleavage and DNA strand exchange occur between bases 3 and 5 (3 out of 10) of the core sequence and the other cleavage and strand exchange occur somewhere between bases 5 and 11 (1 out of 10) (Fig. 2B). Interestingly, in one transconjugant a G nucleotide was found at position 5 in both DR-L and DR-R. In five other transconjugants, a mix of A and G nucleotides was found at position 5 in DR-L or both in DR-L and DR-R (Fig. 2B). The finding of the same base (G) at position 5 in both DR-L and DR-R could be consistent with mismatch repair of single bp substitutions during recombination. Such event has been previously described for the lambda bacteriophage [17]. Furthermore, the mix of bases (A or G) at position 5 in DR-L or both in DR-L and DR-R suggests that in one transconjugant different subpopulations may present different DR-Ls or different DR-Ls and DR-Rs. This finding is consistent with the hypothesis of tandem integration of SGI1 and then different excision events with cleavage and strand exchange upstream or downstream position 5. Thus, SGI1 excision events in tandem arrays could generate for one transconjugant different subpopulations with different DR-Ls or different DR-Ls and DR-Rs.


Secondary chromosomal attachment site and tandem integration of the mobilizable Salmonella genomic island 1.

Doublet B, Golding GR, Mulvey MR, Cloeckaert A - PLoS ONE (2008)

Comparison of the SGI1 18 bp attP, attB, DR-Ls, DR-Rs of S. Typhimurium.(A) Alignment of the attP site of SGI1 and the primary attB site (1st attB) of S. Typhimurium strain LT2. The sequence of direct repeats left (DR-L) and right (DR-R) flanking integrated SGI1 were indicated. (B) Alignment of the attP site of SGI1 and the secondary attB site (2nd attB) of S. Typhimurium strain LT2. The sequence of direct repeats left (DR-L) and right (DR-R) were determined in ten independent ΔthdF mutant S. Typhimurium LT2 SGI1 transconjugants from three mating experiments. (*) indicated identical positions in the attP site and the attB sites. Positions 1, 6, 12, and 18 are indicated below the 1st and 2nd attB sequences. The specific nucleotides for attP are underlined in DR-Ls and DR-Rs. Sites of possible cleavage during the strand exchange are indicated by arrows in attP. Nucleotides in boldface letters represent illegitimate base pair in the DR-L and DR-R sequences. The sequences of DR-L and DR-R of some transconjugants revealed a mix of G and A at position 5 in repeated attempts.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2297512&req=5

pone-0002060-g002: Comparison of the SGI1 18 bp attP, attB, DR-Ls, DR-Rs of S. Typhimurium.(A) Alignment of the attP site of SGI1 and the primary attB site (1st attB) of S. Typhimurium strain LT2. The sequence of direct repeats left (DR-L) and right (DR-R) flanking integrated SGI1 were indicated. (B) Alignment of the attP site of SGI1 and the secondary attB site (2nd attB) of S. Typhimurium strain LT2. The sequence of direct repeats left (DR-L) and right (DR-R) were determined in ten independent ΔthdF mutant S. Typhimurium LT2 SGI1 transconjugants from three mating experiments. (*) indicated identical positions in the attP site and the attB sites. Positions 1, 6, 12, and 18 are indicated below the 1st and 2nd attB sequences. The specific nucleotides for attP are underlined in DR-Ls and DR-Rs. Sites of possible cleavage during the strand exchange are indicated by arrows in attP. Nucleotides in boldface letters represent illegitimate base pair in the DR-L and DR-R sequences. The sequences of DR-L and DR-R of some transconjugants revealed a mix of G and A at position 5 in repeated attempts.
Mentions: In previous studies, the left and right junctions of SGI1 integrated in the last 18 bp of the thdF gene (named 1st attB site in this study) have been sequenced and analyzed [4], [13], [16]. The sequence of the specific recombinational site (SGI1 attP site) of the extrachromosomal circular form of SGI1 has been also previously determined [13]. Integration of SGI1 in its 1st attB site was shown to occur by recombination mediated by the SGI1 integrase between the 18 bp attP site of the circular form and the 18 bp 1st attB site at the 3′ end of the thdF gene [13]. Compared to the SGI1 attP sequence, the 1st attB site of S. Typhimurium strain LT2 presents two nucleotide substitutions at positions 9 and 12 (Fig. 2A). Analysis of DR-L and DR-R in S. Typhimurium DT104 in which SGI1 is integrated in its 1st attB site demonstrated that these two nucleotide substitutions were always found in the DR-R (Fig. 2A). This result suggests that the cleavage and strand exchange occur somewhere upstream the position 9 during SGI1 integration in its 1st attB site (Fig. 2A). The integration of SGI1 in the 2nd attB site was slightly different. For the 10 ΔthdF mutant S. Typhimurium LT2 transconjugants, the sequences of the left and right junctions were determined to analyze the direct repeat sequences flanking SGI1 in this 2nd attB site (Fig. 2B). As shown in Figure 2B, the sequence of the 2nd attB site in the S. Typhimurium LT2 chromosome differs both in length and sequence from the specific SGI1 attP sequence. Compared to the SGI1 attP site, the 2nd attB site is only 14 bp in length and presents three additional substitutions at positions 3, 5, and 15 to the four gap positions (Fig. 2B). The differences in the SGI1 attP site and the 2nd attB site result in different DR-L and DR-R sequences that allow the cleavage sites during recombination between attP and attB to be estimated (Fig. 2B). The sequences of DR-L and DR-R suggest that one cleavage and DNA strand exchange occur between bases 3 and 5 (3 out of 10) of the core sequence and the other cleavage and strand exchange occur somewhere between bases 5 and 11 (1 out of 10) (Fig. 2B). Interestingly, in one transconjugant a G nucleotide was found at position 5 in both DR-L and DR-R. In five other transconjugants, a mix of A and G nucleotides was found at position 5 in DR-L or both in DR-L and DR-R (Fig. 2B). The finding of the same base (G) at position 5 in both DR-L and DR-R could be consistent with mismatch repair of single bp substitutions during recombination. Such event has been previously described for the lambda bacteriophage [17]. Furthermore, the mix of bases (A or G) at position 5 in DR-L or both in DR-L and DR-R suggests that in one transconjugant different subpopulations may present different DR-Ls or different DR-Ls and DR-Rs. This finding is consistent with the hypothesis of tandem integration of SGI1 and then different excision events with cleavage and strand exchange upstream or downstream position 5. Thus, SGI1 excision events in tandem arrays could generate for one transconjugant different subpopulations with different DR-Ls or different DR-Ls and DR-Rs.

Bottom Line: In the absence of thdF, the frequency of transconjugant formation was reduced by around thirty times of magnitude.At this 2(nd)attB site, we found that a significant fraction of SGI1 transconjugants (43% of wild type and 100% of Delta thdF mutant) contained tandem SGI1 arrays.The existence of a secondary attachment site in the Salmonella chromosome has potential implications for the mobility of SGI1, which may integrate in other attachment sites of other bacterial pathogens that do not possess the 1(st) or 2(nd) specific SGI1 attB sites of Salmonella.

View Article: PubMed Central - PubMed

Affiliation: INRA, UR1282, Infectiologie Animale et Santé Publique, Nouzilly, France. Benoit.Doublet@tours.inra.fr

ABSTRACT

Background: The Salmonella genomic island 1 is an integrative mobilizable element (IME) originally identified in epidemic multidrug-resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) DT104. SGI1 contains a complex integron, which confers various multidrug resistance phenotypes due to its genetic plasticity. Previous studies have shown that SGI1 integrates site-specifically into the S. enterica, Escherichia coli, or Proteus mirabilis chromosome at the 3' end of thdF gene (attB site).

Methodology/principal findings: Here, we report the transfer of SGI1 to a Delta thdF mutant of S. Typhimurium LT2. In the absence of thdF, the frequency of transconjugant formation was reduced by around thirty times of magnitude. Through DNA sequencing SGI1 was shown to integrate specifically into a secondary attachment site (2(nd)attB), which is located in the intergenic region between the chromosomal sodB and purR genes. At this 2(nd)attB site, we found that a significant fraction of SGI1 transconjugants (43% of wild type and 100% of Delta thdF mutant) contained tandem SGI1 arrays. Moreover, in wild type S. Typhimurium LT2 transconjugants, SGI1 integrated into both attachment sites, i.e., thdF and sodB-purR. The formation of SGI1 tandem arrays occurred in both specific attB sites. There was heterogeneity in the size of the SGI1 tandem arrays detected in single transconjugant colonies. Some arrays consisted as far as six SGI1s arranged in tandem. These tandem arrays were shown to persist during serial passages with or without antibiotic selection pressure.

Conclusions/significance: The ability of integration into two distinct chromosomal sites and tandem array formation of SGI1 could contribute to its spread and persistence. The existence of a secondary attachment site in the Salmonella chromosome has potential implications for the mobility of SGI1, which may integrate in other attachment sites of other bacterial pathogens that do not possess the 1(st) or 2(nd) specific SGI1 attB sites of Salmonella.

Show MeSH
Related in: MedlinePlus