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

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.

Figure 1. Chromosomal integration sites and tandem arrays of SGI1 in the S. Typhimurium chromosome.

(A) Schematic view of the primary specific integration site (1^st attB) of SGI1 within the 3′ end of the chromosomal thdF gene of different S. Typhimurium strains. The retron sequence downstream the 1^st attB site is only present in S. Typhimurium strains. The integration of SGI1 tandem arrays (2 copies) in its 1^st attB site is represented. Positions of HindIII restriction sites (H) are indicated without respect of bp scale to show the expected sizes of HindIII fragments corresponding to 1^st DR-L, 1^st DR-R, and attP. (B) Schematic view of the secondary integration site (2^nd attB) of SGI1 between the chromosomal sodB and purR genes. The integration of SGI1 tandem arrays (3 copies) in the 2^nd attB site is represented. Positions of BglI restriction sites (B) are indicated without respect of bp scale to show the expected sizes of BglI fragments corresponding to 2^nd DR-L, 2^nd DR-R, and attP. The sequences of 1^st attB, 2^nd attB, attP, DR-Ls, and DR-Rs are indicated.

Figure 2. 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 (1^st 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 (2^nd 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 1^st and 2^nd 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.

Figure 3. Chromosomal junctions of tandemly-arranged SGI1 islands in the S. Typhimurium LT2 chromosome.

(A) Southern blot hybridization with the 364-bp attP SGI1 probe containing part of S044, 18 bp attP site and part of the int gene of HindIII-digested genomic DNAs of wild type S. Typhimurium LT2 SGI1 transconjugants. Lanes 1-4 correspond to different transconjugants with tandem arrays of SGI1 integrated at the 3′ end of thdF and lanes 5-6 to transconjugants with a single SGI1 integrated at this attB site. (B) Southern blot hybridization with the 364-bp attP SGI1 probe of BglI-digested genomic DNA of ΔthdF mutant S. Typhimurium LT2 SGI1 transconjugants. All transconjugants tested showed the same profile with BglI fragments containing DR-L, DR-R, and attP. The molecular sizes of HindIII or BglI fragments containing DR-L, DR-R, and attP are indicated.

Figure 4. Copy numbers of SGI1 tandem arrays in the S. Typhimurium LT2 chromosome.

(A) Macrorestriction analysis by PFGE of genomic DNAs cut by AscI of S. Typhimurium LT2 SGI1 transconjugants. Lanes: 1, S. Albany strain 7205.00 (SGI1 donor strain); 2-7, S. Typhimurium LT2 SGI1 transconjugants; 8-12, ΔthdF mutant S. Typhimurium LT2 SGI1 transconjugants. The molecular sizes in kilobases are indicated to the left of the panel. The numbers indicated to the right of the bands on the restriction patterns (lane 5 and 10) correspond to the copy numbers of SGI1 in each fragment. The size of the AscI fragments observed are consistent with the expected sizes of 2 to 5 SGI1 copies in tandem. (B) Southern blot hybrization with the p1-9 probe of the PFGE-AscI restriction patterns of Figure 4A. The numbers indicated to the right of the panel correspond to the copy numbers of SGI1 in each fragment revealed by the p1-9 probe. The integration sites of SGI1 are indicated under each transconjugant profiles; (+) integrated SGI1, (−) unoccupied attB site, (na) not applicable.

Figure 5. Analysis of the SGI1 attP overlapping region.

The previously described 18-bp sequence of the attP site is underlined. Positions 1, 6, 12, and 18 are indicated below the attP site. The imperfect inverted repeats are indicated by arrows. Sequences of the different SGI1 attB sites are aligned below the SGI1 attP sequence. Nucleotide in boldface letters represented substitutions compared to the attP sequence. The black box represented the putative 7-bp overlap region in which cleavages and strand exchanges occurred.